Methods and agents for the treatment of ocular disease

ABSTRACT

This disclosure relates generally to methods and agents for treating an ocular disease or disorder. More particularly, the present disclosure relates to the use of CD14 antagonist antibodies for treating an ocular disease or disorder.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.17/678,861, filed Feb. 23, 2022, which is a continuation ofInternational Patent Application PCT/AU2021/051173 filed Oct. 7, 2021,which claims priority to Australian Provisional Application No.2020903624 entitled “Methods and agents for the treatment of oculardisease” filed Oct. 7, 2020, the contents of which are hereinincorporated by reference in its entirety for all purposes.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename: LBTI_001_02US_SeqList.ST26, date created: Jan. 26, 2023, file size 44,012 bytes)

FIELD OF THE INVENTION

This disclosure relates generally to methods and agents for treating anocular disease or disorder. More particularly, the present disclosurerelates to the use of CD14 antagonist antibodies for treating an oculardisease or disorder.

BACKGROUND OF THE INVENTION

Ocular diseases cause vision impairment and blindness in millions ofpeople worldwide, significantly affecting sufferers' quality of life.The diseases are complex and have differing and wide-ranging causes, andonly limited therapeutic and management options.

Age-related macular degeneration (AMD) is the leading cause of blindnessin patients over the age of 50. It is characterized by progressivedegeneration of the photoreceptors, outer retina, and retinal pigmentepithelium at the macula. There are two types of AMD: dry and wet AMD.Most AMD starts as the dry type and in 10-20% of individuals, itprogresses to the wet type. Wet AMD frequently causes a rapid and oftensubstantial loss of central vision in patients. In the wet form of AMD,choroidal neovascularization forms and develops into a network ofvessels that may grow under and through the retinal pigment epithelium.As this is accompanied by leakage of plasma and/or hemorrhage into thesubretinal space, severe and sudden loss of central vision may result.While wet AMD affects approximately 10-20% of individuals withage-related macular degeneration, it accounts for approximately 90% ofall cases of severe vision loss from the disease. AMD has been shown tohave no single cause, and likely results from variable contributionsincluding but not limited to age, genetic predisposition, and/orenvironmental factors. In humans, for example, established epidemiologicrisk factors may include but are not limited to cigarette smoking, diet,female sex, Caucasian race, and a family history of AMD.

Pathological myopia (also known as high myopia or degenerative myopia)is another leading cause of visual impairment worldwide and is the mostfrequent cause of visual impairment in Asian countries. It is a form ofsevere and progressive nearsightedness characterized by changes in thefundus of the eye, due to posterior staphyloma and deficient correctedacuity. Other ocular diseases that are significant causes of loss ofvision worldwide include, for example, diabetic retinopathy, hereditaryretinal dystrophies, proliferative vitreoretinopathy, retinopathy ofprematurity, diabetic macular oedema, neovascular glaucoma and Coats'disease.

Anti-VEGF agents (e.g. anti-VEGF antibodies, VEGF Traps, etc.) are nowwidely used in the treatment of ocular diseases such as wet AMD,diabetic retinopathy, diabetic macular edema, pathological myopia(particularly those pathological myopia patients presenting withchoroidal neovascularization (CNV)) and other ocular diseases, andfunction to inhibit neovascularization and angiogenesis. In mostpatients this can reduce the progression of choroidal neovascularizationand vision loss resulting from the downstream effects ofneovascularization, and has been observed to reduce some aspects ofinflammation in animal models. For example anti-VEGFs may modify theretinal damage induced by neovascularization and therefore resolvefollow-on inflammatory activities by immune cells. However, anti-VEGFagents will not block proinflammatory cytokine release from an alreadyactivated immune cell (see e.g. Funk et al., 2010, Retina,30(9):1412-1419). Despite regular (e.g. monthly) injections of anti-VEGFagents, many patients with ocular disease still proceed to lose theirvision due to the development of fibrotic lesions and atrophy. Inwet-AMD some patients continue to loose visual acuity over 5 years evenwith sustained treatment. In addition, some 10-15% of patients don'trespond to anti-VEGF therapies. Other ocular diseases, have no approvedpharmacologic treatment available for use. Accordingly, there remains aneed for additional agents and methods for treating ocular diseases.

SUMMARY OF THE INVENTION

The present invention arises in part from the surprising determinationthat targeting Cluster of Differentiation 14 (CD14), such as byadministration of an anti-CD14 antagonist antibody, is particularlyefficient at reducing not only neovascularization and lesion size, butalso fibrosis in a laser-induced mouse model of choroidalneovascularization (CNV), and is associated with a reduction of vascularendothelial growth factor (VEGF) in the retinal pigment epithelium(RPE). This is in contrast to gold-standard anti-VEGF therapy (e.g.aflibercept, a VEGF-Trap that is the standard of care for many oculardiseases such as AMD), which at high doses inhibits neovascularizationand reduces lesion size but does not inhibit fibrosis and indeed isassociated with an increased expression in the RPE of VEGF isoforms andother genes associated with the formation of extracellular matrix.

Interestingly, the unexpected reduction in fibrosis in the retinafollowing anti-CD14 treatment appears to be through a novel mechanismthat is not related to lesion size (i.e. not related toangiogenesis/neovascularisation): inhibition of fibrosis followingtreatment with anti-CD14 antibody was uniform and irrespective of theinhibition of lesion size, and VEGF protein levels were reduced in theRPE but not the retina, suggesting that this was through antagonism ofthe mCD14 receptors expressed on the RPE and not anti-CD14 attenuationof infiltrating macrophage activity. Thus, the unexpected anti-fibroticactivity of the anti-CD14 antibody following laser-induced CNV isindependent of its anti-inflammatory and anti-angiogenic (i.e.anti-neovascularisation) activity.

The surprising finding by the present inventors that targeting CD14 caninhibit not only neovascularization (or angiogenesis) in a mouse modelof CNV but can also inhibit fibrosis has significant implications forthe treatment of ocular diseases, many of which are characterized byneovascularization and fibrosis and thus will be amenable to treatmentwith a CD14 antagonist antibody. Of particular note is the fact thatcurrent therapies, while able to inhibit neovascularization, do notnecessarily also inhibit fibrosis. Indeed, it has been reported that40-50% of patients with wet AMD go blind due to ocular fibrosis evenwhen treated with anti-VEGF therapies. In the Comparison of Age-relatedMacular Degeneration Treatments Trials (CATT), it was observed thatfollowing a year of anti-VEGF treatment, 32% of patients developedfibrotic scars after a further year, with 42% and 56% developingfibrotic scars after a further 2 and 5 years, respectively, even whilebeing successfully treated for neovascularization (i.e. fibrosis candevelop in this disease state independently of neovascularization).During this period of fibrosis development, visual acuity decreased(Daniel et al., 2018, Ophthalmology 125(7): 1037-1046). Indeed, it hasbeen postulated that treatment with anti-VEGF agents may actuallypromote fibrosis. Treatment with a CD14 antagonist antibody as describedherein to patients that are receiving or have received anti-VEGFtreatment can therefore inhibit the development of fibrosis, as well asthe separate processes of ocular inflammation and neovascularization,and aid in the retention of vision for patients with AMD and otherocular diseases that are characterized by fibrosis, inflammation andneovascularization.

Thus, in one aspect, provided is a method for treating an ocular diseaseor disorder in a subject, comprising, consisting or consistingessentially of administering an effective amount of a CD14 antagonistantibody to a subject with an ocular disease or disorder. In anotheraspect, provided is a method for inhibiting ocular inflammation,neovascularization and/or fibrosis in a subject with an ocular diseaseor disorder, comprising, consisting or consisting essentially ofadministering an effective amount of a CD14 antagonist antibody to asubject with an ocular disease or disorder. The ocular disease may beselected from among, for example, pathological myopia, AMD, diabeticretinopathy, hereditary retinal dystrophies, proliferativevitreoretinopathy, retinopathy of prematurity, diabetic macular oedema,neovascular glaucoma, dry eye, fibrosis associated with glaucomafiltration surgery (GFS), Coats' disease, non-infectious uveitis (NIU),macular telangiectasia (MacTel), cystoid macular edema, birdshotchorioretinopathy, Vogt-Koyanagi-Harada disease, idiopathic multifocalchoroiditis, retinal vasculitis, branched vein retinal occlusions (BRVO)and central vein retinal occlusions (CRVO), polypoidal choroidalvasculopathy, Familial Exudative Vitreoretinopathy (FEVR), Idiopathicretinitis, Vasculitis, Aneurysms, and Neuroretinitis (IRVAN), Doyonehoneycomb retinal dystrophy, and enhanced S-cone syndrome. In furtherexamples, the method comprises first selecting the subject foradministration of the CD14 antagonist antibody on the basis that thesubject is at risk of developing, or has, ocular fibrosis (e.g. retinal,subretinal or epiretinal fibrosis). In some embodiments, the AMD is wetAMD or dry AMD.

In another aspect, provided is a method for treating AMD in a subject,comprising, consisting or consisting essentially of: selecting a subjectwith AMD who is at risk of developing, or has, ocular fibrosis (e.g.retinal, subretinal or epiretinal fibrosis); and administering aneffective amount of a CD14 antagonist antibody to the subject. Inanother aspect, provided is a method for inhibiting ocular fibrosis in asubject with AMD, comprising, consisting or consisting essentially of:selecting a subject with AMD who is at risk of developing, or has,ocular fibrosis (e.g. retinal, subretinal or epiretinal fibrosis); andadministering an effective amount of a CD14 antagonist antibody to thesubject. In some embodiments, the AMD is wet AMD or dry AMD.

In another aspect, provided is a use of a CD14 antagonist antibody forthe preparation of a medicament for treating an ocular disease ordisorder in a subject, or for inhibiting ocular inflammation,neovascularization and/or fibrosis in a subject with an ocular diseaseor disorder. In some examples, the ocular disease or disorder isselected from among pathological myopia, AMD, diabetic retinopathy,hereditary retinal dystrophies, proliferative vitreoretinopathy,retinopathy of prematurity, diabetic macular oedema, neovascularglaucoma, dry eye, fibrosis associated with glaucoma filtration surgery(GFS), Coats' disease, non-infectious uveitis (NIU), maculartelangiectasia (MacTel), cystoid macular edema, birdshotchorioretinopathy, Vogt-Koyanagi-Harada disease, idiopathic multifocalchoroiditis, retinal vasculitis, branched vein retinal occlusions (BRVO)and central vein retinal occlusions (CRVO), polypoidal choroidalvasculopathy, Familial Exudative Vitreoretinopathy (FEVR), Idiopathicretinitis, Vasculitis, Aneurysms, and Neuroretinitis (IRVAN), Doyonehoneycomb retinal dystrophy, and enhanced S-cone syndrome. In furtherexamples, the method comprises first selecting the subject foradministration of the CD14 antagonist antibody on the basis that thesubject is at risk of developing, or has, ocular fibrosis (e.g. retinal,subretinal or epiretinal fibrosis). In some embodiments, the AMD is wetAMD or dry AMD.

In another aspect, provided is a use of a CD14 antagonist antibody forthe preparation of a medicament for treating AMD in a subject identifiedas being at risk of developing, or having, ocular fibrosis. In a furtheraspect, provided is a use of a CD14 antagonist antibody for thepreparation of a medicament for inhibiting ocular inflammation in asubject with AMD identified as being at risk of developing, or having,ocular fibrosis. In some examples, the AMD is wet AMD or dry AMD.

In some embodiments of the methods and uses, the subject has received ananti-VEGF agent for at least 3, 6, 9, 12, 14, 16 or 18 months. Infurther examples, the subject is a non-responder to anti-VEGF agent. Theanti-VEGF agent may be, for example, an anti-VEGF antibody (e.g.brolicizumab, ranibizumab or faricimab), anti-VEGF antibody mimetic(e.g. abicipar pegol), VEGF Trap molecule (e.g. aflibercept orconbercept), soluble fms-like tyrosine kinase-1, or a tyrosine kinaseinhibitor.

In particular embodiments, the subject is determined to have ocularfibrosis by detection of the presence of hyperreflective material (HRM)by optical coherence tomography (OCT).

In other embodiments, the subject is determined to have ocular fibrosisby confirming the presence of fibrotic lesions by visualization usingfluorescein angiography, color fundus photography (CFP) or fundusautofluorescence (FAF).

The CD14 antagonist antibody may be administered by local ocularadministration, systemic administration or topical administration. Insome examples, the CD14 antagonist antibody is administered by ocularimplantation.

In one embodiment, the CD14 antagonist antibody is selected from:

-   -   (i) an antibody that comprises: a) an antibody VL domain, or        antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and        L-CDR3, wherein: L-CDR1 comprises the sequence RASESVDSFGNSFMH        [SEQ ID NO: 7] (3C10 L-CDR1); L-CDR2 comprises the sequence        RAANLES [SEQ ID NO: 8] (3C10 L-CDR2); and L-CDR3 comprises the        sequence QQSYEDPWT [SEQ ID NO: 9] (3C10 L-CDR3); and b) an        antibody VH domain, or antigen binding fragment thereof,        comprising H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises        the sequence SYAMS [SEQ ID NO: 10] (3C10 H-CDR1); H-CDR2        comprises the sequence SISSGGTTYYPDNVKG [SEQ ID NO: 11] (3C10        H-CDR2); and H-CDR3 comprises the sequence GYYDYHY [SEQ ID NO:        12] (3C10 H-CDR3);    -   (ii) an antibody that comprises: a) an antibody VL domain, or        antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and        L-CDR3, wherein: L-CDR1 comprises the sequence RASESVDSYVNSFLH        [SEQ ID NO: 13] (28C5 L-CDR1); L-CDR2 comprises the sequence        RASNLQS [SEQ ID NO: 14] (28C5 L-CDR2); and L-CDR3 comprises the        sequence QQSNEDPTT [SEQ ID NO: 15] (28C5 L-CDR3); and b) an        antibody VH domain, or antigen binding fragment thereof,        comprising H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises        the sequence SDSAWN [SEQ ID NO: 16] (28C5 H-CDR1); H-CDR2        comprises the sequence YISYSGSTSYNPSLKS [SEQ ID NO: 17] (28C5        H-CDR2); and H-CDR3 comprises the sequence GLRFAY [SEQ ID NO:        18] (28C5 H-CDR3);    -   (iii) an antibody that comprises: a) an antibody VL domain, or        antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and        L-CDR3, wherein: L-CDR1 comprises the sequence RASESVDSYVNSFLH        [SEQ ID NO: 13] (IC14 L-CDR1); L-CDR2 comprises the sequence        RASNLQS [SEQ ID NO: 14] (IC14 L-CDR2); and L-CDR3 comprises the        sequence QQSNEDPYT [SEQ ID NO: 27] (IC14 L-CDR3); and b) an        antibody VH domain, or antigen binding fragment thereof,        comprising H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises        the sequence SDSAWN [SEQ ID NO: 16] (IC14 H-CDR1); H-CDR2        comprises the sequence YISYSGSTSYNPSLKS [SEQ ID NO: 17] (IC14        H-CDR2); and H-CDR3 comprises the sequence GLRFAY [SEQ ID NO:        18] (IC14 H-CDR3); and    -   (iv) an antibody that comprises: a) an antibody VL domain, or        antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and        L-CDR3, wherein: L-CDR1 comprises the sequence RASQDIKNYLN [SEQ        ID NO: 19] (18E12 L-CDR1); L-CDR2 comprises the sequence YTSRLHS        [SEQ ID NO: 20] (18E12 L-CDR2); and L-CDR3 comprises the        sequence QRGDTLPWT [SEQ ID NO: 21] (18E12 L-CDR3); and b) an        antibody VH domain, or antigen binding fragment thereof,        comprising H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises        the sequence NYDIS [SEQ ID NO: 22] (18E12 H-CDR1); H-CDR2        comprises the sequence VIWTSGGTNYNSAFMS [SEQ ID NO: 23] (18E12        H-CDR2); and H-CDR3 comprises the sequence GDGNFYLYNFDY [SEQ ID        NO: 24] (18E12 H-CDR3).

In another embodiment, the CD14 antagonist antibody is selected from:

-   -   (i) an antibody comprising:    -   a VL domain that comprises, consists or consists essentially of        the sequence:

[SEQ ID NO: 1] QSPASLAVSLGQRATISCRASESVDSFGNSFMHWYQQKAGQPPKSSIYRAANLESGIPARFSGSGSRTDFTLTINPVEADDVATYFCQQSYEDPWTFG GGTKLGNQ (3C10 VL);

-   -    and    -   a VH domain that comprises, consists or consists essentially of        the sequence:

[SEQ ID NO: 2] LVKPGGSLKLSCVASGFTFSSYAMSWVRQTPEKRLEWVASISSGGTTYYPDNVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGYYDYHYWGQG TTLTVSS (3C10 VH);

-   -   (ii) an antibody comprising:    -   a VL domain that comprises, consists or consists essentially of        the sequence:

[SEQ ID NO: 3] QSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYCCQQSNEDPTTFG GGTKLEIK (28C5 VL);

-   -    and    -   a VH domain that comprises, consists or consists essentially of        the sequence:

[SEQ ID NO: 4] LQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSA (28C5 VH);

(iii) an antibody comprising: a VL domain that comprises, consists orconsists essentially of the sequence:

[SEQ ID NO: 25] QSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFG GGTKLEIK (IC14 VL);

-   -    and    -   a VH domain that comprises, consists or consists essentially of        the sequence:

[SEQ ID NO: 26] LQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSS (IC14 VH);

-   -    and    -   (iv) an antibody comprising:    -   a VL domain that comprises, consists or consists essentially of        the sequence:

[SEQ ID NO: 5] QTPSSLSASLGDRVTISCRASQDIKNYLNWYQQPGGTVKVLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQRGDTLPWTFGGGTKL EIK (18E12 VL);

-   -    and    -   a VH domain that comprises, consists or consists essentially of        the sequence:

[SEQ ID NO: 6] LESGPGLVAPSQSLSITCTVSGFSLTNYDISWIRQPPGKGLEWLGVIWTSGGTNYNSAFMSRLSITKDNSESQVFLKMNGLQTDDTGIYYCVRGDGNFYLYNFDYWGQGTTLTVSS (18E12 VH).

In some examples, the CD14 antagonist antibody is humanized or chimeric.In a particular example, the CD14 antagonist antibody comprises:

-   -   a light chain comprising the amino acid sequence

[SEQ ID NO: 32] DIVLTQSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

-   -    and    -   a heavy chain comprising the amino acid sequence:

[SEQ ID NO: 33] DVQLQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K.

The CD14 antagonist antibody may be administered in combination with, orformulated for use in combination with, an ancillary agent. In someexamples, the CD14 antagonist antibody and the ancilliary agent areadministered simultaneously or sequentially. In particular examples, theancillary agent is selected from among an anti-VEGF agents, andangiotensin converting enzyme (ACE) inhibitor, a connective tissuegrowth factor (CTGF) inhibitor, a complement inhibitor (e.g. a C3, C5,factor B, factor D, or properdin inhibitor), an angiopoietin 2 (Ang-2)inhibitor, a PDGF inhibitor, a statin, and a steroid. The anti-VEGFagent may be selected from among an anti-VEGF antibody (e.g.brolicizumab, ranibizumab or faricimab), anti-VEGF antibody mimetic(e.g. abicipar pegol), VEGF Trap molecule (e.g. aflibercept orconbercept), soluble fms-like tyrosine kinase-1, and a tyrosine kinaseinhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the disclosure are described herein, by way ofnon-limiting example only, with reference to the following drawings.

FIG. 1 is a graphical representation of angiogenesis seven days afterlaser induced choroidal neovascularization. (A) Graph of mean+SEM lesionsize in eyes treated with different concentrations of aflibercept (15ug/μl, 60 ug/μl or 80 μg/ml), vehicle or anti-VEGF164 neutralizingantibody (300 ng/ul). (B) Graph of mean±SEM lesion size in eyes treatedwith aflibercept (80 ul/ml), anti-CD14 or vehicle. One-way ANOVA,post-hoc Tukey's test, ** p<0.01, n≥8.

FIG. 2 is a graphical representation of fibrosis 7 days after laserinduced choroidal neovascularization. Graph of mean±SEM ratio offibrosis in vehicle and anti-CD14 treated eyes evaluated at day 7.One-way ANOVA, post-hoc Tukey's test, *p<0.05; ** p<0.01, n≥8.

FIG. 3 is a graphical representation of the correlation between fibrosisand angiogenesis in eyes treated with anti-CD14 and vehicle 7 days afterlaser induced choroidal neovascularization.

FIG. 4 is a graphical representation of VEGF isoform expression in RPEisolated from eyes treated with PBS, anti-CD14 or aflibercept (80 μg/μl)seven days after laser induced CNV. (A) Vegf120 gene expression (copiesVegf120/copies of Hprt). (B) Vegf120 gene expression (copiesVegf120/copies of Gapdh). (C) Vegf164 gene expression (copiesVegf164/copies of Hprt). (D) Vegf164 gene expression (copiesVegf164/copies of Gapdh). (E) Vegf188 gene expression (copiesVegf188/copies of Hprt). (F) Vegf188 gene expression (copiesVegf188/copies of Gapdh).

FIG. 5 is a graphical representation of Tgfb1 and Col1a1 expression inRPE isolated from eyes treated with PBS, anti-CD14 or aflibercept (80μg/μl) seven days after laser induced CNV. (A) Tgfb1 gene expression(copies Tgfb1/copies of Hprt). (B) Tgfb1 gene expression (copiesTgfb1/copies of Gapdh). (C) Col1a1 gene expression (copies Col1a1/copiesof Hprt). (D) Col1a1 gene expression (copies Col1a1/copies of Gapdh).(One way ANOVA, Posthoc Tukeys,*p<0.05, **p<0.01; ***p<0.0001).

FIG. 6 is a graphical representation of VEGF expression in the retinaand RPE at 2, 7 and 14 days after laser induced CNV with treatment withanti-CD14 antibody. (A) Graph of mean+SEM (and individual data) for VEGFexpression in the retina. (B) Graph of mean+SEM (and individual data)for VEGF expression in the RPE. (N=6 per group; Two-way ANOVA; posthocTukeys test,*p<0.01).

FIG. 7 is a graphical representation of the recruitment of CD45+leukocytes population and subpopulations (A-F) across differenttreatments at two days post-CNV. Data are representative of fourindependent experiments. Each population is represented as a percentageof single live cells. N=7-8 per group, mean±SEM. One-way ANOVA, *P<0.05,**P<0.01,***P<0.001.

FIG. 8 is a graphical representation of the recruitment of CD45+leukocytes population and subpopulations (A-F) across differenttreatments at 7 days post-CNV as assessed by flow cytometry. Data arerepresentative of two independent experiments. N=4-8 per group,mean±SEM. One-way ANOVA, *P<0.05, **P<0.01,****P<0.0001.

FIG. 9 is a graphical representation of the recruitment of CD45+leukocytes population and subpopulations (A-F) post-CNV across the 7-daytime course for each treatment. Data are representative of sixindependent experiments. N=4-8 per group, mean±SEM. Two-way ANOVA,*P<0.05, **P<0.01,****P<0.0001.

FIG. 10 is a graphical representation of the recruitment of leucocytes,and the proportion of leucocytes that is represented by myeloid cells,dendritic cells, inflammatory monocytes, resident monocytes/macrophages,and neutrophils present within RPE/choroids 7 days after laser inducedCNV. (A) Changes in the recruitment of CD45+/CD34+/CCR2+/MHCII+ cells(fibrocytes) two days after laser induced CNV. (B) Change inCD45+/CD34+/CCR2+/MHCII+ cells (fibrocytes) seven days after laserinduced CNV. (C) Comparison of CD45+/CD34+/CCR2+/MHCII+ cells expressedas a percentage of live cells. Data are representative of sixindependent experiments. N=4-8 per group, mean±SEM. Two-way ANOVA,*P<0.05, **P<0.01, ****P<0.0001.

FIG. 11 is a graphical representation of the changes in the CD14+CD11b+myeloid population and the expression of CD14 in CD11b+ myeloid cellsubpopulations (A-E) across different treatments at 2 days post-CNV.Data are representative of four independent experiments. N=7-8 pergroup, mean±SEM. One-way ANOVA, *P<0.05, **P<0.01,****P<0.0001.

FIG. 12 is a graphical representation of CD11b+ myeloid cellsubpopulations (A-E) across different treatments at 7 days post-CNV(A-E). Data are representative of two independent experiments. N=4 pergroup, mean±SEM. One-way ANOVA, *P<0.05, **P<0.01,***P<0.001.

FIG. 13 is a graphical representation of changes in the CD14+CD11b+myeloid population and the expression of CD14 in CD11b+ myeloid cellsubpopulations (A-E) post-CNV across the 7-day time course for eachtreatment. Data are representative of six independent experiments. N=4-8per group, mean±SEM. Two-way ANOVA, *P<0.05, **P<0.01,***P<0.001.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (or).

The terms “active agent” and “therapeutic agent” are usedinterchangeably herein and refer to agents that prevent, reduce orameliorate at least one symptom of a disease or disorder.

The terms “administration concurrently” or “administering concurrently”or “co-administering” and the like refer to the administration of asingle composition containing two or more agents, or the administrationof each agent as separate compositions and/or delivered by separateroutes either contemporaneously or simultaneously or sequentially withina short enough period of time that the effective result is equivalent tothat obtained when all such agents are administered as a singlecomposition. By “simultaneously” is meant that the agents areadministered at substantially the same time, and desirably together inthe same formulation. By “contemporaneously” it is meant that the agentsare administered closely in time, e.g., one agent is administered withinfrom about one minute to within about one day before or after another.Any contemporaneous time is useful. However, it will often be the casethat when not administered simultaneously, the agents will beadministered within about one minute to within about eight hours andsuitably within less than about one to about four hours. Whenadministered contemporaneously, the agents are suitably administered atthe same site on the subject. The term “same site” includes the exactlocation, but can be within about 0.5 to about 15 centimeters,preferably from within about 0.5 to about 5 centimeters. The term“separately” as used herein means that the agents are administered at aninterval, for example at an interval of about a day to several weeks ormonths. The agents may be administered in either order. The term“sequentially” as used herein means that the agents are administered insequence, for example at an interval or intervals of minutes, hours,days or weeks. If appropriate the agents may be administered in aregular repeating cycle.

The term “antagonist antibody” is used in the broadest sense, andincludes an antibody that inhibits or decreases the biological activityof an antigen to which the antibody binds (e.g., CD14). For example, anantagonist antibody may partially or completely block interactionbetween a receptor (e.g., CD14) and a ligand (e.g., a DAMP or PAMP), ormay practically decrease the interaction due to tertiary structurechange or down regulation of the receptor. Thus, a CD14 antagonistantibody encompasses antibodies that bind to CD14 and that block,inhibit, nullify, antagonize, suppress, decrease or reduce (includingsignificantly), in any meaningful degree, a CD14 agonist activity,including activation of downstream pathways such as Toll-like receptor(TLR) signaling pathways (e.g., TLR4 signaling pathway) and theTIR-domain-containing adapter-inducing IFN-β (TRIF) pathway, orelicitation of a cellular response (e.g., production of pro-inflammatorymediators including pro-inflammatory cytokines) to CD14 binding by aCD14 ligand (e.g., a DAMP or PAMP).

The term “antibody” herein is used in the broadest sense andspecifically covers naturally occurring antibodies, monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), antibody fragments, or any other antigen-bindingmolecule so long as they exhibit the desired immuno-interactivity. Anaturally occurring “antibody” includes within its scope animmunoglobulin comprising at least two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region iscomprised specific CH domains (e.g., CH1, CH2 and CH3). Each light chainis comprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementarydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs arranged from amino-terminus to carboxy-terminusin the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Theconstant regions of the antibodies may mediate the binding of animmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system. The antibodies can be of any isotype(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2), subclass or modified version thereof (e.g., IgG1isotype, which carries L234A and L235A double mutations (IgG1-LALA)).The antibodies can be of any species, chimeric, humanized or human. Inother embodiments, the antibody is a homomeric heavy chain antibody(e.g., camelid antibodies) which lacks the first constant region domain(CH1) but retains an otherwise intact heavy chain and is able to bindantigens through an antigen-binding domain. The variable regions of theheavy and light chains in the antibody-modular recognition domain (MRD)fusions will contain a functional binding domain that interacts with anantigen of interest.

The “variable domain” (variable domain of a light chain (VL), variabledomain of a heavy chain (VH)) as used herein denotes each of the pair oflight and heavy chain domains which are involved directly in binding theantibody to the antigen. The variable light and heavy chain domains havethe same general structure and each domain comprises four FRs whosesequences are widely conserved, connected by three CDRs or“hypervariable regions”. The FRs adopt a β-sheet conformation and theCDRs may form loops connecting the β-sheet structure. The CDRs in eachchain are held in their three-dimensional structure by the FRs and formtogether with the CDRs from the other chain the antigen binding site.

The term “antigen-binding portion” when used herein refer to the aminoacid residues of an antibody which are responsible for antigen-bindinggenerally, which generally comprise amino acid residues from the CDRs.Thus, “CDR” or “complementarity determining region” (also referred to as“hypervariable region”) are used interchangeably herein to refer to theamino acid sequences of the light and heavy chains of an antibody whichform the three-dimensional loop structure that contributes to theformation of an antigen binding site. There are three CDRs in each ofthe variable regions of the heavy chain and the light chain, which aredesignated “CDR1”, “CDR2”, and “CDR3”, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region that binds the antigen. The exactboundaries of these CDRs have been defined differently according todifferent systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as “Kabat CDRs”. Chothiaand coworkers (Chothia and Lesk, 1987. J. Mol. Biol. 196: 901-917;Chothia et al., 1989. Nature 342: 877-883) found that certainsub-portions within Kabat CDRs adopt nearly identical peptide backboneconformations, despite having great diversity at the level of amino acidsequence. These sub-portions were designated as “L1”, “L2”, and “L3”, or“H1”, “H2”, and “H3”, where the “L” and the “H” designate the lightchain and the heavy chain regions, respectively. These regions may bereferred to as “Chothia CDRs”, which have boundaries that overlap withKabat CDRs. Other boundaries defining CDRs overlapping with the KabatCDRs have been described by Padlan (1995. FASEB J. 9: 133-139) andMacCallum (1996. J. Mol. Biol. 262(5): 732-745). Still other CDRboundary definitions may not strictly follow one of these systems, butwill nonetheless overlap with the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding.

As used herein, the term “framework region” or “FR” refers to theremaining sequences of a variable region minus the CDRs. Therefore, thelight and heavy chain variable domains of an antibody comprise from N-to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRsand FRs are typically determined according to the standard definition ofKabat, E. A., et al., Sequences of Proteins of Immunological Interest,5th ed., Public Health Service, National Institutes of Health, Bethesda,Md. (1991) and/or those residues from a “hypervariable loop”.

As used herein, the terms “light chain variable region” (“VL”) and“heavy chain variable region” (VH) refer to the regions or domains atthe N-terminal portion of the light and heavy chains respectively whichhave a varied primary amino acid sequence for each antibody. Thevariable region of an antibody typically consists of the amino terminaldomain of the light and heavy chains as they fold together to form athree-dimensional binding site for an antigen. Several subtypes of VHand VL, based on structural similarities, have been defined, for exampleas set forth in the Kabat database.

The term “chimeric antibody” refers to antibodies that comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Thus, the FRs and CDRs of a humanized antibody neednot correspond precisely to the parental (i.e., donor) sequences, e.g.,a donor antibody CDR or the consensus framework may be mutagenized bysubstitution, insertion, and/or deletion of at least one amino acidresidue so that the CDR or FR at that site does not correspond to eitherthe donor antibody or the consensus framework. Typically, suchmutations, however, will not be extensive and will generally avoid “keyresidues” involved in binding to an antigen. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (see, for example, Winnaker, FromGenes to Clones (Verlagsgesellschaft, Weinheim, 1987)). A “consensusimmunoglobulin sequence” may thus comprise a “consensus frameworkregion(s)” and/or a “consensus CDR(s)”. In a family of immunoglobulins,each position in the consensus sequence is occupied by the amino acidoccurring most frequently at that position in the family. If two aminoacids occur equally frequently, either can be included in the consensussequence. In general, the humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will generally comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al. (1986. Nature321:522-525), Riechmann et al. (1988. Nature 332:323-329) and Presta(1992. Curr. Op. Struct. Biol. 2:593-596). A humanized antibody can beselected from any class of immunoglobulins, including IgM, IgG, IgD,IgA, and IgE, and any isotype, including without limitation IgG1, IgG2,IgG3, and IgG4. A humanized antibody may comprise sequences from morethan one class or isotype, and particular constant domains may beselected to optimize desired effector functions using techniques wellknown in the art. As used herein, the term “key residue” refers tocertain residues within the variable region that have more impact on thebinding specificity and/or affinity of an antibody, in particular ahumanized antibody. A key residue includes, but is not limited to, oneor more of the following: a residue that is adjacent to a CDR, apotential glycosylation site (can be either N- or O-glycosylation site),a rare residue, a residue capable of interacting with the antigen, aresidue capable of interacting with a CDR, a canonical residue, acontact residue between heavy chain variable region and light chainvariable region, a residue within the Vernier zone, and a residue in theregion that overlaps between the Chothia definition of a variable heavychain CDR1 and the Kabat definition of the first heavy chain framework.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992. J. Mol. Biol. 224: 487-499).Vernier zone residues form a layer underlying the CDRs and may impact onthe structure of CDRs and the affinity of the antibody.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (1987. J. Mol. Biol. 196: 901-917; 1992. J.Mol. Biol. 227: 799-817), both are incorporated herein by reference).According to Chothia et al., critical portions of the CDRs of manyantibodies have nearly identical peptide backbone confirmations despitegreat diversity at the level of amino acid sequence. Each canonicalstructure specifies primarily a set of peptide backbone torsion anglesfor a contiguous segment of amino acid residues forming a loop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs to an “acceptor antibody”. In someembodiments, the donor antibody is an antibody from a species differentfrom the antibody from which the FRs are obtained or derived. In thecontext of a humanized antibody, the term “donor antibody” refers to anon-human antibody providing one or more CDRs.

As used herein, the terms “acceptor” and “acceptor antibody” refer to anantibody providing at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or 100% of the amino acid sequences of one or more ofthe FRs. In some embodiments, the term “acceptor” refers to the antibodyamino acid sequence providing the constant region(s). In otherembodiments, the term “acceptor” refers to the antibody amino acidsequence providing one or more of the FRs and the constant region(s). Inspecific embodiments, the term “acceptor” refers to a human antibodyamino acid sequence that provides at least 80%, preferably, at least85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acidsequences of one or more of the FRs. In accordance with this embodiment,an acceptor may contain at least 1, at least 2, at least 3, least 4, atleast 5, or at least 10 amino acid residues that does (do) not occur atone or more specific positions of a human antibody. An acceptorframework region and/or acceptor constant region(s) may be, for example,derived or obtained from a germline antibody gene, a mature antibodygene, a functional antibody (e.g., antibodies well-known in the art,antibodies in development, or antibodies commercially available).

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The terms “heavy chain variable region CDR1” and “H-CDR1” are usedinterchangeably, as are the terms “heavy chain variable region CDR2” and“H-CDR2”, the terms “heavy chain variable region CDR3” and “H-CDR3”, theterms “light chain variable region CDR1” and “L-CDR1”; the terms “lightchain variable region CDR2” and “L-CDR2” and the terms “light chainvariable region CDR3” and “L-CDR3” antibody fragment. Throughout thespecification, complementarity determining regions (“CDR”) are definedaccording to the Kabat definition unless specified otherwise. The Kabatdefinition is a standard for numbering the residues in an antibody andit is typically used to identify CDR regions (Kabat et al., (1991), 5thedition, NIH publication No. 91-3242).

Antigen binding can be performed by “fragments” or “antigen-bindingfragments” of an intact antibody. Herein, both terms are usedinterchangeably. Examples of binding fragments encompassed within theterm “antibody fragment” of an antibody include a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; an Fd fragmentconsisting of the VH and CH1 domains; an Fv fragment consisting of theVL and VH domains of a single arm of an antibody; a single domainantibody (dAb) fragment (Ward et al., 1989. Nature 341:544-546), whichconsists of a VH domain; and an isolated complementary determiningregion (CDR). In a particular embodiment, the antibody of the presentdisclosure is an antigen-binding fragment that lacks all or a portion ofthe Fc region.

A “single chain variable Fragment (scFv)” is a single protein chain inwhich the VL and VH regions pair to form monovalent molecules (known assingle chain Fv (scFv); see, e.g., Bird et al., 1988. Science242:423-426; and Huston et al., 1988. Proc. Natl. Acad. Sci.85:5879-5883). Although the two domains VL and VH are coded for byseparate genes, they can be joined, using recombinant methods, by anartificial peptide linker that enables them to be made as a singleprotein chain. Such single chain antibodies include one or more antigenbinding moieties. These antibody fragments are obtained usingconventional techniques known to those of skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

The term “monoclonal antibody” and abbreviations “MAb” and “mAb”, asused herein, refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a single antigen.Furthermore, in contrast to polyclonal antibody preparations thattypically include different antibodies directed against differentdeterminants (epitopes), each mAb is directed against a singledeterminant on the antigen. The modifier “monoclonal” is not to beconstrued as requiring production of the antibody by any particularmethod. Monoclonal antibodies may be produced, for example, by a singleclone of antibody-producing cells, including hybridomas. The term“hybridoma” generally refers to the product of a cell-fusion between acultured neoplastic lymphocyte and a primed B- or T-lymphocyte whichexpresses the specific immune potential of the parent cell.

An antibody “that binds” an antigen of interest (e.g., CD14) is one thatbinds the antigen with sufficient affinity such that the antibody isuseful as a therapeutic agent in targeting a cell or tissue expressingthe antigen, and does not significantly cross-react with other proteins.In such embodiments, the extent of binding of the antibody to a“non-target” protein will be less than about 10% of the binding of theantibody, oligopeptide or other organic molecule to its particulartarget protein as determined, for example, by fluorescence activatedcell sorting (FACS) analysis, enzyme-linked immunosorbent assay (ELISA),immunoprecipitation or radioimmunoprecipitation (RIA). Thus, an antibodythat antagonizes CD14 suitably inhibits or decreases production ofpro-inflammatory mediators, including pro-inflammatorycytokines/chemokines. With regard to the binding of an antibody to atarget molecule, the term “specific binding” or “specifically binds to”or is “specific for” a particular polypeptide or an epitope on aparticular polypeptide target means binding that is measurably differentfrom a non-specific interaction. Specific binding can be measured, forexample, by determining binding of a molecule compared to binding of acontrol molecule, which generally is a molecule of similar structurethat does not have binding activity. For example, specific binding canbe determined by competition with a control molecule that is similar tothe target, for example, an excess of non-labeled target. In this case,specific binding is indicated if the binding of the labeled target to aprobe is competitively inhibited by excess unlabeled target. Thespecific region of the antigen to which the antibody binds is typicallyreferred to as an “epitope”. The term “epitope” broadly includes thesite on an antigen which is specifically recognized by an antibody orT-cell receptor or otherwise interacts with a molecule. Generallyepitopes are of active surface groupings of molecules such as aminoacids or carbohydrate or sugar side chains and generally may havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. As will be appreciated by one of skillin the art, practically anything to which an antibody can specificallybind could be an epitope.

An “anti-VEGF agent” (or VEGF inhibitor or VEGF antagonist) refers to amolecule capable of neutralizing, blocking, inhibiting, abrogating,reducing or interfering with an activity of vascular endothelial growthfactor (VEGF), including but not limited to its binding to one or moreVEGF receptors. Anti-VEGF agents include anti-VEGF antibodies includingantigen-binding fragments thereof, receptor molecules and derivativeswhich bind specifically to VEGF thereby sequestering its binding to oneor more receptors (e.g. VEGF Traps), anti-VEGF receptor antibodies andVEGF receptor antagonists such as small molecule inhibitors of the VEGFRtyrosine kinases, and fusions proteins. By way of example, exemplaryanti-VEGF agents include the anti-VEGF antibodies bevacizumab andranibizumab, the VEGF Trap aflibercept (also referred to as VEGF TrapEye), and small molecules such as lapatinib, sunitinib, sorafenib,axitinib, and pazopanib that inhibit the tyrosine kinases stimulated byVEGF. “Anti-VEGF therapy” refers to a treatment regimen using ananti-VEGF agent.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. Thus, use of the term “comprising” and the likeindicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present. By“consisting of” is meant including, and limited to, whatever follows thephrase “consisting of”. Thus, the phrase “consisting of” indicates thatthe listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they affect theactivity or action of the listed elements.

By “effective amount”, in the context of treating a disease or conditionis meant the administration of an amount of an agent or composition toan individual in need of such treatment or prophylaxis, either in asingle dose or as part of a series, that is effective for the preventionof incurring a symptom, holding in check such symptoms, and/or treatingexisting symptoms, of that condition. The effective amount will varydepending upon the age, health and physical condition of the individualto be treated and whether symptoms of disease are apparent, thetaxonomic group of individual to be treated, the formulation of thecomposition, the assessment of the medical situation, and other relevantfactors. Optimal dosing schedules can be calculated from measurements ofdrug accumulation in the body of the subject. Optimum dosages may varydepending on the relative potency in an individual subject, and cangenerally be estimated based on EC50 values found to be effective in invitro and in vivo animal models. Persons of ordinary skill can easilydetermine optimum dosages, dosing methodologies and repetition rates. Itis expected that the amount will fall in a relatively broad range thatcan be determined through routine trials.

The terms “inhibit” or “reduce” and the like in relation to ocularinflammation, neovascularization or fibrosis or loss of vision refers toat least a small but measurable inhibition or reduction in ocularinflammation, neovascularization or fibrosis or loss of vision of asubject with an ocular disease following administration of an anti-CD14antagonist antibody, compared to in the absence of administration of theantibody. Typically, the inhibition or decrease is a statisticallysignificant inhibition or decrease. In some embodiments, ocularinflammation, neovascularization or fibrosis or loss of vision isdecreased or inhibited by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or more. In one example, loss of vision is assessed by assessingthe subject's best corrected visual acuity (BCVA), as is known in theart, e.g. using standard Early Treatment of Diabetic Retinopathy Study(ETDRS)-like charts). In other examples, the extent ofneovascularization and/or fibrosis is assess using standard techniquessuch as fluorescein angiography, color fundus photography, opticalcoherence tomography or fundus autofluorescence.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state.

The term “ligand”, as used herein, refers to any molecule which iscapable of binding a receptor.

The term “local ocular administration” refers to non-systemicintraocular administration and includes, for example, intravitreal,subconjunctival, subretinal, retrobulbar and/or intracameraladministration. Local ocular administration can be achieved by discreteinjection, such as with a needle or other delivery device, or byimplantation, such as for sustained or extended release of the activeagent.

The term “non-responder to an anti-VEGF agent” refers to a subject withan ocular disease who does not, or would not, respond in the expectedpositive manner to therapy with an anti-VEGF agent. A non-respondertherefore includes subjects who have, or would have, an incompleteresponse, poor response or no response to an anti-VEGF agent.Non-responders can be identified and classified according to standardcriteria and definitions accepted by those skilled in the art. In someexamples, a non-responder is defined as a subject exhibiting with nogain (e.g. no change or a loss (e.g. more than 0.2 or 5 letters) inbest-corrected visual acuity (BVCA) after a period (e.g. 3, 6 or 12months) of anti-VEGF treatment compared to baseline (i.e. at initiationof treatment), and/or a subject with persistent sub- or intra-retinalfluid, as detected optical coherence tomography (OCT) scan, at 3 monthsafter initiation of anti-VEGF treatment (see e.g. Gao et al., 2020,Scientific Reports, 10:1341). In other examples, biomarkers are used tocategorize a subject as a non-responder. For example, serum levels ofglycerophosphocholine (GPC), lysophosphatidylcholine (LysoPC) and/orphosphatidylserine (PS) can be used to predict whether a subject is anon-responder to an anti-VEGF agent (Gao et al., 2020, ScientificReports, 10:1341). In such examples, the subject may not have begunanti-VEGF therapy, but is categorized as a non-responder on the basisthat they are predicted to not respond to anti-VEGF therapy due to theirbiomarker profile.

By “pharmaceutically acceptable carrier” is meant a pharmaceuticalvehicle comprised of a material that is not biologically or otherwiseundesirable, i.e., the material may be administered to a subject alongwith the selected active agent without causing any or a substantialadverse reaction. Carriers may include excipients and other additivessuch as diluents, detergents, coloring agents, wetting or emulsifyingagents, pH buffering agents, preservatives, transfection agents and thelike.

Similarly, a “pharmacologically acceptable” salt, ester, amide, prodrugor derivative of a compound as provided herein is a salt, ester, amide,prodrug or derivative that this not biologically or otherwiseundesirable.

The terms “polynucleotide,” “genetic material,” “genetic forms,”“nucleic acids” and “nucleotide sequence” include RNA, cDNA, genomicDNA, synthetic forms and mixed polymers, both sense and antisensestrands, and may be chemically or biochemically modified or may containnon-natural or derivatized nucleotide bases, as will be readilyappreciated by those skilled in the art.

The term “pro-inflammatory mediator” means an immunoregulatory agentthat favors inflammation. Such agents include, cytokines such aschemokines, interleukins (IL), lymphokines, and tumor necrosis factor(TNF) as well as growth factors. In specific embodiments, thepro-inflammatory mediator is a “pro-inflammatory cytokine”. Typically,pro-inflammatory cytokines include IL-1α, IL-1β, IL-6, and TNF-α, whichare largely responsible for early responses. Other pro-inflammatorymediators include LIF, IFN-γ, IFN-β, IFN-α, OSM, CNTF, TGF-β, GM-CSF,TWEAK, IL-11, IL-12, IL-15, IL-17, IL-1β, IL-19, IL-20, IL-8, IL-16,IL-22, IL-23, IL-31 and IL-32 (Tato et al., 2008. Cell 132:900; Cell132:500, Cell 132:324). Pro-inflammatory mediators may act as endogenouspyrogens (IL-1, IL-6, IL-17, TNF-α), up-regulate the synthesis ofsecondary mediators and pro-inflammatory cytokines by both macrophagesand mesenchymal cells (including fibroblasts, epithelial and endothelialcells), stimulate the production of acute phase proteins, or attractinflammatory cells. In specific embodiments, the term “pro-inflammatorycytokine” relates to TNF-α, IL-1 α, IL-6, IFNβ, IL-1β, IL-8, IL-17 andIL-18.

As used herein, the term “ocular disease or disorder” refers to adisease or disorder that affects the eye, manifesting in ocularinflammation, neovascularization and/or fibrosis, and ultimately a lossof vision. Exemplary ocular diseases or disorders that are suitable fortreatment in accordance with the methods and agents of the presentdisclosure include wet age-related macular degeneration (wet AMD),pathological myopia, diabetic retinopathy, hereditary retinaldystrophies (also referred to as inherited retinal dystrophies (IRD),e.g. Leber's congenital amaurosis (LCA), Stargardt macular dystrophy,Best disease and Bestrophinopathies, Juvenile X-linked retinoschisis,Congenital stationary night blindness (CSNB), Achromatopsia and Ushersyndrome), dry AMD, proliferative vitreoretinopathy, retinopathy ofprematurity, diabetic macular oedema, neovascular glaucoma, dry eye,fibrosis associated with glaucoma filtration surgery (GFS), Coats'disease, non-infectious uveitis (NIU), macular telangiectasia (MacTel),cystoid macular edema, birdshot chorioretinopathy, Vogt-Koyanagi-Haradadisease, idiopathic multifocal choroiditis, retinal vasculitis, branchedvein retinal occlusions (BRVO) and central vein retinal occlusions(CRVO), polypoidal choroidal vasculopathy, Familial ExudativeVitreoretinopathy (FEVR), Idiopathic retinitis, Vasculitis, Aneurysms,and Neuroretinitis (IRVAN), Doyone honeycomb retinal dystrophy, andenhanced S-cone syndrome.

As used herein, the term “systemic administration” or “administeredsystemically” or “systemically administered” means introducing an agentinto a subject outside of the central nervous system. As such, it isclear that local ocular administration as well as ocular implantationare not within the scope of the terms “systemic administration”,“administered systemically” or “systemically administered”. An agent(e.g. an antibody) or pharmaceutical composition as described herein canbe systemically administered in any acceptable form such as in a tablet,liquid, capsule, powder, or the like; by intravenous, intraperitoneal,intramuscular, subcutaneous or parenteral injection; by transdermaldiffusion or electrophoresis; and by minipump or other implantedextended release device or formulation. According to some embodiments,systemic administration is carried out by a route selected from thegroup consisting of intraperitoneal, intravenous, subcutaneous andintranasal administration, and combinations thereof.

The terms “subject”, “patient” and “individual” used interchangeablyherein, refer to any subject, particularly a vertebrate subject, andeven more particularly a mammalian subject, (e.g. human) with a MI.

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect in asubject in need of treatment, that is, a subject who has a oculardisease or disorder. By “treatment” is meant ameliorating or preventingone or more symptoms or effects (e.g. consequences) of a ocular diseaseor disorder. In particular examples, treatment includes ameliorating orpreventing ocular inflammation, neovascularization and/or fibrosis,and/or reducing loss of vision. Reference to “treatment”, “treat” or“treating” does not necessarily mean to reverse or prevent any or allsymptoms or effects of a ocular disease or disorder. For example, thesubject may ultimately suffer one or more symptoms or effects, but thenumber and/or severity of the symptoms or effects is reduced and/or thequality of life is improved compared to prior to treatment.

Each embodiment described herein is to be applied mutatis mutandis toeach and every embodiment unless specifically stated otherwise.

-   -   2. CD14 Antagonist Antibodies

The present disclosure provides methods, uses and compositions thatinclude a CD14 antagonist antibody for treating an ocular disease ordisorder in a subject. The present disclosure also provides methods,uses and compositions that include a CD14 antagonist antibody fortreating an ocular disease or disorder.

The present disclosure contemplates any CD14 antagonist antibody thatbinds to CD14, such as human CD14 (e.g. human mCD14 or sCD14) and blocksthe binding of a DAMP or PAMP to CD14 and/or that binds to CD14 andinhibits or decreases a CD14 agonist-mediated response resulting in theproduction of pro-inflammatory mediators, including the production ofpro-inflammatory cytokines. In some embodiments, a CD14 antagonistantibody of the present invention inhibits binding of a CD14 agonist,suitably a DAMP or PAMP, to CD14 thus inhibiting or decreasing theproduction of pro-inflammatory cytokines. In illustrative examples ofthis type, the CD14 antagonist antibody is selected from the 3C10antibody that binds an epitope comprised in at least a portion of theregion from amino acid 7 to amino acid 14 of human CD14 (van Voohris etal., 1983. J. Exp. Med. 158: 126-145; Juan et al., 1995. J. Biol. Chem.270(29): 17237-17242), the MEM-18 antibody that binds an epitopecomprised in at least a portion of the region from amino acid 57 toamino acid 64 of CD14 (Bazil et al., 1986. Eur. J. Immunol.16(12):1583-1589; Juan et al., 1995. J. Biol. Chem. 270(10): 5219-5224),the 4C1 antibody (Adachi et al., 1999. J. Endotoxin Res. 5: 139-146;Tasaka et al., 2003. Am. J. Respir. Cell. Mol. Biol.; 2003.29(2):252-258), as well as the 28C5 and 23G4 antibodies that inhibitbinding of LPS and suppress production of pro-inflammatory cytokines,and the 18E12 antibody that partly inhibits binding of LPS andsuppresses production of pro-inflammatory cytokines (U.S. Pat. Nos.5,820,858, 6,444,206 and 7,326,569 to Leturcq et al.). In someembodiments, a CD14 antagonist antibody of the present disclosureinhibits binding of CD14 to a TLR such as TLR4, thereby blockingCD14-agonist mediated response, illustrative examples of which includethe F1024 antibody disclosed in International Publication WO2002/42333.Other CD14 antagonist antibodies include the single-chain antibodyscFv2F9 and the related human-mouse chimeric antibody Hm2F9 (Tang et al.2007, Immunopharmacol Immunotoxicol 29,375-386; and Shen et al., 2014,DNA Cell Biol. 33(9): 599-604). Further examples of CD14 antagonistantibodies include the anti-human CD14 18D11 IgG1 mAb, 18D11 IgG1F(ab)′2 fragment and the chimeric r18D11 antibody (IgG 2/4) (see e.g.Lau et al., 2013, J Immunol 191:4769-4777). Each of the above referencesrelating to CD14 antagonist antibodies is incorporated herein byreference in its entirety. The CD14 antagonist antibody may be afull-length immunoglobulin antibody or an antigen-binding fragment of anintact antibody, representative examples of which include a Fabfragment, a F(ab′)2 fragment, an Fd fragment consisting of the VH andCH1 domains, an Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, a single domain antibody (dAb) fragment (Wardet al., 1989. Nature 341:544-546), which consists of a VH domain; and anisolated CDR. Suitably, the CD14 antagonist antibody is a chimeric,humanized or human antibody.

In some embodiments, the CD14 antagonist antibody comprises a VH and VLof an antibody disclosed in U.S. Pat. No. 5,820,858:

-   -   (1) an antibody comprising:        a VL domain comprising, consisting or consisting essentially of        the sequence:

[SEQ ID NO: 1]QSPASLAVSLGQRATISC RASESVDSFGNSFMH WYQQKAGQPPKSSIY RAANLESGIPARFSGSGSRTDFTLTINPVEADDVATYFC QQSYEDPWT FGGGTKLGNQ (3C10 VL);anda VH domain comprising, consisting or consisting essentially of thesequence:

[SEQ ID NO: 2]LVKPGGSLKLSCVASGFTFS SYAMS WVRQTPEKRLEWVA SISSGGTTYYPDNVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCAR GYYDYHY WGQGTTLTVSS (3C10 VH);

-   -   (2) an antibody comprising:        a VL domain comprising, consisting or consisting essentially of        the sequence:

[SEQ ID NO: 3]QSPASLAVSLGQRATISC RASESVDSYVNSFLH WYQQKPGQPPKLLIY RASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYCC QQSNEDPTT FGGGTKLEIK (28C5 VL);anda VH domain comprising, consisting or consisting essentially of thesequence:

[SEQ ID NO: 4]LQQSGPGLVKPSQSLSLTCTVTGYSIT SDSAWN WIRQFPGNRLEWMG YISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVR GLRFAY WGQGTLVTVSA (28C5 VH);and

-   -   (3) an antibody comprising:        a VL domain comprising, consisting or consisting essentially of        the sequence:

[SEQ ID NO: 5] QTPSSLSASLGDRVTISC RASQDIKNYLN WYQQPGGTVKVLIY YTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFC QRGDTLPWT FGGGTKLEIK (18E12 VL);anda VH domain comprising, consisting or consisting essentially of thesequence:

[SEQ ID NO: 6]LESGPGLVAPSQSLSITCTVSGFSLT NYDIS WIRQPPGKGLEWLG VIWTSGGTNYNSAFMSRLSITKDNSESQVFLKMNGLQTDDTGIYYCVR GDGNFYLYNFDY WGQGTTLTVSS (18E12 VH);

Also contemplated are antibodies that comprise the VL and VH CDRsequences of the above antibodies and related antibodies, representativeembodiments of which include:

-   -   (1) an antibody that comprises: a) an antibody VL domain, or        antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and        L-CDR3, wherein: L-CDR1 comprises the sequence RASESVDSFGNSFMH        [SEQ ID NO: 7] (3C10 L-CDR1); L-CDR2 comprises the sequence        RAANLES [SEQ ID NO: 8] (3C10 L-CDR2); and L-CDR3 comprises the        sequence QQSYEDPWT [SEQ ID NO: 9] (3C10 L-CDR3); and b) an        antibody VH domain, or antigen binding fragment thereof,        comprising H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises        the sequence SYAMS [SEQ ID NO: 10] (3C10 H-CDR1); H-CDR2        comprises the sequence SISSGGTTYYPDNVKG [SEQ ID NO: 11] (3C10        H-CDR2); and H-CDR3 comprises the sequence GYYDYHY [SEQ ID NO:        12] (3C10 H-CDR3);    -   (2) an antibody that comprises: a) an antibody VL domain, or        antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and        L-CDR3, wherein: L-CDR1 comprises the sequence RASESVDSYVNSFLH        [SEQ ID NO: 13] (28C5 L-CDR1); L-CDR2 comprises the sequence        RASNLQS [SEQ ID NO: 14] (28C5 L-CDR2); and L-CDR3 comprises the        sequence QQSNEDPTT [SEQ ID NO: 15] (28C5 L-CDR3); and b) an        antibody VH domain, or antigen binding fragment thereof,        comprising H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises        the sequence SDSAWN [SEQ ID NO: 16] (28C5 H-CDR1); H-CDR2        comprises the sequence YISYSGSTSYNPSLKS [SEQ ID NO: 17] (28C5        H-CDR2); and H-CDR3 comprises the sequence GLRFAY [SEQ ID NO:        18] (28C5 H-CDR3);    -   (3) an antibody that comprises: a) an antibody VL domain, or        antigen binding fragment thereof, comprising L-CDR1, L-CDR2 and        L-CDR3, wherein: L-CDR1 comprises the sequence RASESVDSYVNSFLH        [SEQ ID NO: 13] (IC14 L-CDR1); L-CDR2 comprises the sequence        RASNLQS [SEQ ID NO: 14] (IC14 L-CDR2); and L-CDR3 comprises the        sequence QQSNEDPYT [SEQ ID NO: 27] (IC14 L-CDR3); and b) an        antibody VH domain, or antigen binding fragment thereof,        comprising H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises        the sequence SDSAWN [SEQ ID NO: 16] (IC14 H-CDR1); H-CDR2        comprises the sequence YISYSGSTSYNPSLKS [SEQ ID NO: 17] (IC14        H-CDR2); and H-CDR3 comprises the sequence GLRFAY [SEQ ID NO:        18] (IC14 H-CDR3); and    -   (4) an antibody that comprises: a) an antibody VL domain, or        antigen binding fragment thereof, comprising an L-CDR1, L-CDR2        and L-CDR3, wherein: L-CDR1 comprises the sequence RASQDIKNYLN        [SEQ ID NO: 19] (18E12 L-CDR1); L-CDR2 comprises the sequence        YTSRLHS [SEQ ID NO: 20] (18E12 L-CDR2); and L-CDR3 comprises the        sequence QRGDTLPWT [SEQ ID NO: 21] (18E12 L-CDR3); and b) an        antibody VH domain, or antigen binding fragment thereof,        comprising H-CDR1, H-CDR2 and H-CDR3, wherein: H-CDR1 comprises        the sequence NYDIS [SEQ ID NO: 22] (18E12 H-CDR1); H-CDR2        comprises the sequence VIWTSGGTNYNSAFMS [SEQ ID NO: 23] (18E12        H-CDR2); and H-CDR3 comprises the sequence GDGNFYLYNFDY [SEQ ID        NO: 24] (18E12 H-CDR3).

In some embodiments, the CD14 antagonist antibody is humanized. Inillustrative examples of this type, the humanized CD14 antagonistantibodies suitably comprise a donor CDR set corresponding to a CD14antagonist antibody (e.g., one of the CD14 antagonist antibodiesdescribed above), and a human acceptor framework. The human acceptorframework may comprise at least one amino acid substitution relative toa human germline acceptor framework at a key residue selected from thegroup consisting of: a residue adjacent to a CDR; a glycosylation siteresidue; a rare residue; a canonical residue; a contact residue betweenheavy chain variable region and light chain variable region; a residuewithin a Vernier zone; and a residue in a region that overlaps between aChothia-defined VH CDR1 and a Kabat-defined first heavy chain framework.Techniques for producing humanized mAbs are well known in the art (see,for example, Jones et al., 1986. Nature 321: 522-525; Riechmann et al.1988. Nature 332:323-329; Verhoeyen et al., 1988. Science 239:1534-1536; Carter et al., 1992. Proc. Natl. Acad. Sci. USA 89:4285-4289; Sandhu, J S., 1992. Crit. Rev. Biotech. 12: 437-462, andSinger et al., 1993. J. Immunol. 150: 2844-2857). A chimeric or murinemonoclonal antibody may be humanized by transferring the mouse CDRs fromthe heavy and light variable chains of the mouse immunoglobulin into thecorresponding variable domains of a human antibody. The mouse frameworkregions (FR) in the chimeric monoclonal antibody are also replaced withhuman FR sequences. As simply transferring mouse CDRs into human FRsoften results in a reduction or even loss of antibody affinity,additional modification might be required in order to restore theoriginal affinity of the murine antibody. This can be accomplished bythe replacement of one or more human residues in the FR regions withtheir murine counterparts to obtain an antibody that possesses goodbinding affinity to its epitope. See, for example, Tempest et al. (1991.Biotechnology 9:266-271) and Verhoeyen et al. (1988 supra). Generally,those human FR amino acid residues that differ from their murinecounterparts and are located close to or touching one or more CDR aminoacid residues would be candidates for substitution.

In one embodiment, the CD14 antagonist antibody is the IC14 antibody(Axtelle et al., 2001. J. Endotoxin Res. 7: 310-314; and U.S. Pat. Appl.No. 2006/0121574, which are incorporated herein by reference in theirentirety) or an antigen-binding fragment thereof. The IC14 antibody is achimeric (murine/human) monoclonal antibody that specifically binds tohuman CD14. IC14 was derived from the murine 28C5 noted above (see, U.S.Pat. Nos. 5,820,858, 6,444,206 and 7,326,569 to Leturcq et al., andLeturcq et al., 1996. J. Clin. Invest. 98: 1533-1538). Thus, in oneexample, the CD14 antagonist antibody comprises the VL domain and a VHdomain, wherein:

-   -   the VL domain comprises the amino acid sequence:

[SEQ ID NO: 25] QSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFG GGTKLEIK;

-   -    and    -   the VH domain comprises the amino acid sequence:

[SEQ ID NO: 26] LQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLR FAYWGQGTLVTVSS;

-   -   or    -   the VL domain comprises the amino acid sequence:

[SEQ ID NO: 30] DIVLTQSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNED PYTFGGGTKLEIK;

-   -   the VH domain comprises the amino acid sequence:

[SEQ ID NO: 31] DVQLQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVR GLRFAYWGQGTLVTVSS.

-   -    and

In another example, the CD14 antagonist antibody comprises light chainand heavy chain of IC14, wherein:

-   -   the light chain comprises the amino acid sequence:

[SEQ ID NO: 28] QSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC;

-   -    and    -   the heavy chain comprises the amino acid sequence:

[SEQ ID NO: 29] LQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

-   -   or    -   the light chain comprises the amino acid sequence:

[SEQ ID NO: 32] DIVLTQSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

-   -    and    -   the heavy chain comprises the amino acid sequence:

[SEQ ID NO: 33] DVQLQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K.

Additional antagonist antibodies of CD14 suitable for use in the methodsherein can be identified by methods well known to those skilled in theart. These methods generally comprise determining whether an antibody iscapable of directly antagonizing CD14. For example, the methods mayinvolve determining whether an antibody is capable of inhibiting ordecreasing the amount or agonist activity of CD14, wherein the abilityto inhibit or decrease the amount or agonist activity of CD14 indicatesthat the antibody may be suitable for use in treating MI. In someembodiments, the antibody is contacted with CD14, or a cell thatexpresses CD14 on its surface, or a nucleic acid sequence from whichCD14 is expressed, suitably in the presence of a CD14 agonist such as aDAMP or PAMP, wherein a decrease in the amount or agonist activity ofCD14 in the presence of the agonist, when compared to a control,indicates that the antibody binds to CD14 and directly antagonizes CD14.A decrease or inhibition of CD14 agonist activity, includes for exampleinhibiting, or decreasing activation of, downstream pathways such as TLRsignaling pathways (e.g., TLR4 signaling pathway) and the TRIF pathway,or elicitation of a cellular response (e.g., production ofpro-inflammatory mediators including pro-inflammatory cytokines).

These methods may be carried out in vivo, ex vivo or in vitro. Inparticular, the step of contacting an antibody with CD14 or with a cellthat expresses CD14 on its surface (e.g., immune cells) may be carriedout in vivo, ex vivo or in vitro. The methods may be carried out in acell-based or a cell-free system. For example, the method may comprise astep of contacting a cell expressing CD14 on its surface with anantibody and determining whether the contacting of the cell with theantibody leads to a decrease in the amount or agonist activity of CD14.In such a cell-based assay, the CD14 and/or the antibody may beendogenous to the host cell, may be introduced into a host cell ortissue, may be introduced into the host cell or tissue by causing orallowing the expression of an expression construct or vector or may beintroduced into the host cell by stimulating or activating expressionfrom an endogenous gene in the cell. In such a cell-based method, theamount of activity of CD14 may be assessed in the presence or absence ofan antibody in order to determine whether the agent is altering theamount of CD14 in the cell, such as through regulation of CD14expression in the cell or through destabilization of CD14 protein withinthe cell, or altering the CD14 agonist activity of the cell. Thepresence of a lower CD14 agonist activity or a decreased amount of CD14on the cell surface in the presence of the antibody indicates that theantibody may be a suitable antagonist of CD14 for use in accordance withthe present disclosure.

In some examples, it is further determined whether the antibody lackssubstantial or detectable binding to another cellular component,suitably a binding partner of CD14, such as a CD14 binding partner thatis either secreted (e.g., MD2) or located on the cell membrane (e.g.,TLR4), to thereby determine that the antibody is a specific antagonistof CD14. In a non-limiting example of this type, the antibody iscontacted in the presence of a CD14 agonist such as a DAMP or PAMP (1)with a wild-type cell that expresses CD14 on its surface (e.g., animmune cell such a macrophage), and (2) with a CD14 negative cell (e.g.,an immune cell that is the same as in (1) but has a loss of function inthe CD14 gene). If the antibody inhibits a CD14 agonist activity of thewild-type cell but not of the CD14 negative cell, this indicates thatthe antibody is a CD14 specific antagonist. Cells of this type may beconstructed using routine procedures or animals.

In other examples, potential CD14 antagonist antibodies are assessed invivo, such as, for example, in an animal model. In such an in vivomodel, the effects of the antibody may be assessed in the circulation(e.g., blood) or heart, or in other organs such as the eye, lung, liver,kidney, or the brain. In particular examples, models of ocular diseasesare used to assess the activity of the antibody (e.g. CNV laser mousemodel).

Exemplary antagonist antibodies of CD14 effect a decrease in CD14activity or levels of at least 5%, at least 10%, at least 25%, at least50%, at least 60%, at least 75%, or at least 85% or more compared to inthe absence of the antibody. In some examples, the antibody may resultin a decrease in CD14 agonist activity or levels such that the agonistactivity or level of CD14 is no longer detectable in the presence of theantibody. Such a decrease may be seen in the sample being tested or, forexample where the method is carried out in an animal model.

Preferably, the antibody is a specific antagonist of CD14 as describedabove. However, this does not mean that a specific antagonist of CD14has a complete absence of off-target antagonistic activity. In thisregard, the specific antagonist of CD14 may have negligible or a minordirect binding and effect on other cellular components, such that theantagonism of the activity, signaling or expression of a non-CD14cellular component, is less than less than 15%, less than 10%, less than5%, less than 1%, or less than 0.1% of the direct binding and effect ofthat agent on the activity, signaling or expression of CD14.

Levels or amounts of CD14 may be measured by assessing expression of theCD14 gene. Gene expression may be assessed by looking at mRNA productionor levels or at protein production or levels. Expression products suchas mRNA and proteins may be identified or quantified by methods known inthe art. Such methods may utilize hybridization to specifically identifythe mRNA of interest. For example such methods may involve PCR orreal-time PCR approaches. Methods to identify or quantify a protein ofinterest may involve the use of antibodies that bind that protein. Forexample, such methods may involve western blotting. Regulation of CD14gene expression may be compared in the presence and absence of anantibody. Thus, antibodies can be identified that decrease CD14 geneexpression compared to the level seen in the absence of the antibody.Such antibodies may be suitable antagonists of CD14 in accordance withthe present disclosure.

The methods for identifying suitable antagonist antibodies for use inaccordance with the present disclosure may assess the agonist activityof CD14. For example, such a method may be carried out using peripheralblood mononuclear cells. Such cells will produce cytokines such as IL-1α, IL-6, TNF-α, IFN-β, IL-1β, IL-17 and IL-8 on response to stimulationwith, for example, LPS. Methods may therefore comprise combiningperipheral blood mononuclear cells with the antibody or a vehicle andadding LPS. The cells may then be incubated for an amount of time (e.g.,24 hours) to allow the production of pro-inflammatory mediators such ascytokines. The level of cytokines such as IL-1α, IL-6, TNF-α, IFN-β,IL-1β, IL-17 and IL-8 produced by the cells in that time period can thenbe assessed. If the antibody has anti-CD14 properties, then theproduction of such cytokines should be reduced compared to thevehicle-treated cells.

In some examples, the CD14 antagonist antibody is a multifunctional(e.g. bifunctional) antibody. In some examples, the bifunctionalantibody is specific for CD14 and an inflammatory molecule and/orangiogenic molecule and/or fibrogenic molecule, such that thebifunctional antibody inhibits the activity of the inflammatory moleculeand/or angiogenic and/or fibrogenic molecule. For example, thebifunctional antibody may be specific for CD14 and a complement protein(e.g. C3, C5, factor B, factor D, or properdin), angiopoietin 2 (Ang-2),VEGF or platelet-derived growth factor (PDGF).

3. Ancillary Agents and Interventions

The CD14 antagonist antibody may administered alone or in combinationwith other active agents (also referred to as “ancillary agents”) orother interventions, such as agents and interventions useful for thetreatment of an ocular disease or disorder.

Ancillary agents suitable for the purposes of the present disclosureinclude, for example, anti-VEGF agents, angiotensin converting enzyme(ACE) inhibitors, connective tissue growth factor (CTGF) inhibitors,complement inhibitors (including C3, C5, C5, factor B, factor D, andproperdin inhibitors), angiopoietin 2 (Ang-2) inhibitors, PDGFinhibitors, statins, and steroids.

In one example, the ancillary agent is an anti-VEGF agent (or VEGFinhibitor or antagonist). VEGF is a well-characterized signal proteinthat stimulates angiogenesis. Anti-VEGF agents have been developed forthe purposes of inhibiting angiogenesis (or neovascularization), andhave become one of the few approved pharmacologic treatments for oculardiseases that are characterized by neovascularization (e.g. CNV).Anti-VEGF agents are typically administered intravitreally, althoughcould be administered by other ocular routes, such as other local ocularroutes or even topically (e.g. in the form of eye drops) with theappropriate formulation, or using extended or sustained release systemsor implants. Anti-VEGF agents include, for example, anti-VEGF antibodies(e.g. brolicizumab, ranibizumab or faricimab (a bifunctional antibodythat binds both VEGF and Ang-2), VEGF Trap molecules (e.g. afliberceptand conbercept), other antagonistic VEGF-binding molecules such assoluble fms-like tyrosine kinase-1 (sFlt-1 or sVEGFR-1) and antibodymimetics such as abicipar pegol (a designed ankyrin repeat protein;DARPin), and tyrosine kinase inhibitors. In some examples, the anti-VEGFagent is delivered using a vector, such as a viral vector, whereby thevector contains nucleic acid encoding the anti-VEGF agent (e.g.ADVM-022, which is an adeno-associated virus (AAV) vector encodingaflibercept; RGX-314, which is an AAV vector encoding an anti-VEGF Fab;and rAAV.sFLT-1, an AAV vector encoding sFlt-1).

In another example, administration of the antibody is in conjunctionwith an intervention, such as laser photocoagulation or photodynamictherapy.

When combination therapy is desired, the CD14 antagonist antibody isadministered separately, simultaneously or sequentially with one or moreancillary agents or interventions. In some embodiments, this may beachieved by administering, such as systemically, a single composition orpharmacological formulation that includes both types of agent, or byadministering two separate compositions or formulations at the sametime, wherein one composition includes the CD14 antagonist antibody andthe other the ancillary agent. In other embodiments, the treatment withthe CD14 antagonist antibody may precede or follow the treatment withthe ancillary agent by intervals ranging from minutes to hours or evendays or weeks.

In some situations, the antibody and ancillary agent are administeredwithin about 1-12 hours of each other or within about 2-6 hours of eachother. In other situations, it may be desirable to extend the timeperiod for treatment significantly, where one or more days (e.g. 1, 2,3, 4, 5, 6, 7 or 8 days) or weeks (e.g. 1, 2, 3, 4, 5, 6, 7 or 8 weeks)lapse between the respective administrations. In embodiments where theancillary agent is administered separately to the CD14 antagonistantibody, it will be understood that the ancillary agent can beadministered by a method which is the same or different to that of theadministration method used for the CD14 antagonist antibody.

Where two or more agents are administered to a subject “in conjunction”or “concurrently” they may be administered in a single composition atthe same time, or in separate compositions at the same time, or inseparate compositions separated in time.

4. Compositions

As described herein, the use of a CD14 antagonist antibody, whetheralone or in combination with one or more ancillary agents, can treat anocular disease; and/or can inhibit ocular inflammation,neovascularization and/or fibrosis in a subject with an ocular disease.The CD14 antagonist antibody and optionally the ancillary agent can beadministered either by themselves or with a pharmaceutically acceptablecarrier. Thus, also provided herein are compositions comprising a CD14antagonist antibody for use in treating an ocular disease or disorder;and/or inhibiting ocular inflammation, neovascularization and/orfibrosis, in a subject with an ocular disease or disorder.

The CD14 antagonist antibodies may be formulated in a conventionalmanner using one or more pharmaceutically acceptable carriers,stabilizers or excipients (vehicles) to form a pharmaceuticalcomposition as is known in the art, in particular with respect toprotein active agents. Carrier(s) are “acceptable” in the sense of beingcompatible with the other ingredients of the composition and notdeleterious to the recipient (e.g. patient) thereof. Suitable carrierstypically include physiological saline or ethanol polyols such asglycerol or propylene glycol.

The antibody may be formulated as neutral or salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups) and which are formed with inorganicacids such as hydrochloric or phosphoric acids, or such organic acidssuch as acetic, oxalic, tartaric and maleic. Salts formed with the freecarboxyl groups may also be derived from inorganic bases such as sodium,potassium, ammonium, calcium, or ferric hydroxides, and organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine andprocaine.

The compositions may be suitably formulated for systemic administration,including intravenous, intramuscular, subcutaneous, or intraperitonealadministration, local ocular administration (e.g. intravitreal,subconjunctival, subretinal, retrobulbar or intracameraladministration), or topical delivery (e.g. as eye drops), andconveniently comprise sterile aqueous solutions of the antibody, whichare preferably isotonic with the targeted environment in the recipient.Such formulations are typically prepared by dissolving solid activeingredient in water containing physiologically compatible substancessuch as sodium chloride, glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be prepared in unit ormulti-dose containers, for example, sealed ampoules or vials.

The compositions may incorporate a stabilizer, such as for examplepolyethylene glycol, proteins, saccharides (for example trehalose),amino acids, inorganic acids and admixtures thereof. Stabilizers areused in aqueous solutions at the appropriate concentration and pH. ThepH of the aqueous solution is adjusted to be within the range of5.0-9.0, preferably within the range of 6-8. In formulating theantibody, anti-adsorption agent may be used. Other suitable excipientsmay typically include an antioxidant such as ascorbic acid.

The compositions may be formulated as controlled release preparationswhich may be achieved through the use of polymer to complex or absorbthe proteins. Appropriate polymers for controlled release formulationsinclude for example polyester, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, and methylcellulose. Another possible method forcontrolled release is to incorporate the antibody into particles of apolymeric material such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively,instead of incorporating these agents into polymeric particles, it ispossible to entrap these materials in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Thus, in particular examples, the CD14 antagonist antibody andoptionally an ancillary agent are formulated for sustained or extendedrelease, such as in non-biodegradable implants (e.g. the PosteriorMicropump™ (PMP) drug delivery system by Replenish, Inc. (Humayun et al.2014, Transl Vis Sci Technol. 3(6):5), and the port delivery system(PDS) by Genentech (Campochiaro et al. 2019, Opthamology,126:1141-1154), biodegradable implants, nanoformulations (includingnanoparticuales, microparticles, liposomes and dendrimers) and hydrogels(see e.g. Seah et al., 2020, Eye, 34:1341-1356). In some examples, wherethe composition is formulated for topical administration to the eye(e.g. as eye drops), the composition contains cell-penetrating peptides(CPPs) to assist in penetration of the CD14 antagonist antibody acrosstissue barriers, both into cells and between cells (see e.g. Cogan etal., 2017, Investigative Ophthalmology & Visual Science, 58:2578-2590).Modes and compositions for delivery of therapeutic antibodies to the eyeare well known in the art and any one can be used for administration ofa CD14 antagonist antibody in accordance with the present disclosure(Mandel et al., 2018, Adv Drug Deliv Rev. 126: 67-95).

A CD14 antagonist antibody and optionally an ancillary agent may also beadministered directly to the airways in the form of an aerosol. For useas aerosols, the inhibitors of the present invention in solution orsuspension may be packaged in a pressurized aerosol container togetherwith suitable propellants, for example, hydrocarbon propellants likepropane, butane, or isobutane with conventional adjuvants. The materialsof the present invention also may be administered in a non-pressurizedform such as in a nebulizer or atomizer.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

5. Methods of Treatment

The present disclosure provides for therapeutic methods for treating asubject with an ocular disease or disorder, such as an ocular disease ordisorder that is characterized by ocular inflammation,neovascularization and/or fibrosis (i.e. ocular inflammation,neovascularization and/or fibrosis is typically observed in patientswith the ocular disease or disorder). In particular examples, the oculardisease or disorder is characterized by the presence or development offibrosis, and treatment of the subject prevents, inhibits or amelioratesfibrosis.

Exemplary ocular diseases or disorders include, but are not limited to,pathological myopia, AMD (including wet and dry AMD), diabeticretinopathy, hereditary retinal dystrophies, proliferativevitreoretinopathy, retinopathy of prematurity, diabetic macular oedema,neovascular glaucoma, dry eye, fibrosis associated with glaucomafiltration surgery (GFS), Coats' disease, non-infectious uveitis (NIU),macular telangiectasia (MacTel), cystoid macular edema, birdshotchorioretinopathy, Vogt-Koyanagi-Harada disease, idiopathic multifocalchoroiditis, retinal vasculitis, branched vein retinal occlusions (BRVO)and central vein retinal occlusions (CRVO), polypoidal choroidalvasculopathy, Familial Exudative Vitreoretinopathy (FEVR), Idiopathicretinitis, Vasculitis, Aneurysms, and Neuroretinitis (IRVAN), Doyonehoneycomb retinal dystrophy, and enhanced S-cone syndrome. Such oculardiseases or disorders are characterized by ocular (e.g. retinal,subretinal, epiretinal, choroidal, corneal, optical nerve, macula, etc.)inflammation, neovascularization and/or fibrosis. Consequently,administration of a CD14 antagonist antibody, which is demonstratedherein to inhibit both neovascularization and fibrosis and is a knownanti-inflammatory agent, can have particular therapeutic benefits.Typically, the subject is diagnosed with the ocular disease or disorderprior to application of the methods of the present disclosure, such asby a medical practitioner on the basis of accepted criteria known tothose practitioners and widely used in the art.

In one example, the ocular disease or disorder is pathological myopia(also known as high myopia or degenerative myopia). Pathological myopiais a form of severe and progressive nearsightedness characterized bychanges in the fundus of the eye, due to posterior staphyloma anddeficient corrected acuity, and is commonly defined as having aspherical equivalent ≤−6.0D, or axial length ≥26.5 mm (Shih et al.,2006, Br J Ophthalmol 90: 546-550). Clinically, the funduscopic changesassociated with pathological myopia include straightened and stretchedvessels, temporal peripapillary atrophic crescent, tilting of the opticdisc, posterior staphyloma, lacquer cracks in the Bruch's membrane,subretinal haemorrhage and CNV, which is associated with a significantreduction in corrected vision. Thus, in some embodiments, the subjectwith pathological myopia has CNV. Pathological myopia is alsocharacterized by the development of ocular fibrosis, including aftertreatment with anti-VEGF agents (see e.g., Ahn et al., 2016, Retina,36(11):2140-2149; Xiao et al., 2020, Br J Ophthalmol; and Zhu et al.2016. Cochrane Database of Systematic Reviews. Issue 12. Art. No.:CD011160).

In a further example, the ocular disease is AMD, which is characterizedby neovascularisation causing vision distortion and thickening of theBruch's membrane. In one embodiment, the AMD is dry AMD. Dry AMD ischaracterized by the accumulation of small drusen in the retinal pigmentepithelium (RPE) leading to a loss of RPE and to a degeneration of theretina in the macular region. In some instances, the dry AMD is earlydry AMD. In other instances, the dry AMD is late dry AMD withoutgeographic atrophy. In other examples, the AMD is wet AMD. Wet AMD isusually preceded by dry AMD and is characterized by neovascularmembranes derived from the choroidal vasculature that invade Bruch'smembrane, leak, and often cause detachments of the RPE and or the neuralretina. These new capillaries are abnormally permeable, allowingaccumulation of serum and blood under the RPE and or the neurosensoryretina. The leaking blood vessels generally lead to a scarring of themacula. The loss of vision in patients with wet AMD can be rapid andresult in functional blindness. In patients with wet AMD the complexformed by the choroidal new vessels and the fibrous tissue can destroythe photoreceptors relatively rapidly, e.g. within 3-24 months.

In another example, the ocular disease is diabetic retinopathy. Diabeticretinopathy is an ocular complication of diabetes, characterized bymicroaneurysms, hard exudates, hemorrhages, and venous abnormalities inthe non-proliferative form and neovascularization, preretinal orvitreous hemorrhages, and fibrovascular proliferation in theproliferative form. Hyperglycemia induces these microvascular retinalchanges, leading to blurred vision, dark spots or flashing lights, andsudden loss of vision. Like pathological myopia and AMD, diabeticretinopathy is characterized by ocular inflammation, neovascularizationand fibrosis (see e.g. Roy et al., 2016, Experimental Eye Research142:71e75; Murakami et al., 2020, Progress in Retinal and Eye Research74:100778).

In other examples, the ocular disease is hereditary retinal dystrophy,proliferative vitreoretinopathy, retinopathy of prematurity, diabeticmacular oedema, neovascular glaucoma, dry eye, fibrosis associated withGFS, Coats' disease, non-infectious uveitis, macular telangiectasia,cystoid macular edema, birdshot chorioretinopathy, Vogt-Koyanagi-Haradadisease, idiopathic multifocal choroiditis, retinal vasculitis, branchedvein retinal occlusions and central vein retinal occlusions, polypoidalchoroidal vasculopathy, FEVR, IRVAN, Doyone honeycomb retinal dystrophy,or enhanced S-cone syndrome, each of which are also characterized byocular inflammation, neovascularization and/or fibrosis (see e.g. Idreeset al., 2019, Int Ophthalmol Clin. 59(1): 221-240, Enriquez et al. 2020,Asia-Pacific Journal of Ophthalmology 9(4):358-368; Cetin et al. 2018,Graefe's Archive for Clinical and Experimental Ophthalmology,256:1801-1806, Marano et al. 2000, Graefe's Arch Clin Exp Ophthalmol,238:760-764; Sen et al., 2019, Indian J Ophthalmol. 67(6): 763-771;Schlunck et al. 2016, Exp. Eye Res. 142, 76-82; Zhavoronkov et al.,2016, Cell Cycle 15(15): 2087; Golzarri et al., 2020, Ocul ImmunolInflamm Oct. 6; 1-5; Khojasteh et al., 2021, J Ophthalmol Apr.30:6674290; Alsalamah et al., 2021, Ophthalmol Retina 5(9):918-927;Tarib et al., 2020, J Clin Exp Ophthal, 11(1):1000824).

Contemplated herein are therefore methods for treating an oculardisease, such as an ocular disease associated with or characterized byocular inflammation, neovascularization and/or fibrosis (e.g.pathological myopia, AMD (including wet and dry AMD), diabeticretinopathy, hereditary retinal dystrophies, proliferativevitreoretinopathy, retinopathy of prematurity, diabetic macular oedema,neovascular glaucoma, dry eye, fibrosis associated with GFS, Coats'disease, non-infectious uveitis, macular telangiectasia, cystoid macularedema, birdshot chorioretinopathy, Vogt-Koyanagi-Harada disease,idiopathic multifocal choroiditis, retinal vasculitis, branched veinretinal occlusions and central vein retinal occlusions, polypoidalchoroidal vasculopathy, FEVR, IRVAN, Doyone honeycomb retinal dystrophy,or enhanced S-cone syndrome), in a subject by administering to thesubject a CD14 antagonist antibody, and optionally administering anancillary agent or performing an intervention, and methods forinhibiting ocular inflammation, neovascularization and/or fibrosis in asubject with an ocular disease (e.g. pathological myopia, AMD (includingwet and dry AMD), diabetic retinopathy, hereditary retinal dystrophies,proliferative vitreoretinopathy, retinopathy of prematurity, diabeticmacular oedema, neovascular glaucoma, fibrosis associated with GFS,Coats' disease, non-infectious uveitis, macular telangiectasia, cystoidmacular edema, birdshot chorioretinopathy, Vogt-Koyanagi-Harada disease,idiopathic multifocal choroiditis, retinal vasculitis, branched veinretinal occlusions and central vein retinal occlusions, polypoidalchoroidal vasculopathy, FEVR, IRVAN, Doyone honeycomb retinal dystrophy,or enhanced S-cone syndrome) by administering to the subject a CD14antagonist antibody, and optionally administering an ancillary agent orperforming an intervention. Where the method is for treating fibrosisassociated with GFS, the subject has had GFS. Thus, provided are methodsfor inhibiting ocular inflammation, neovascularization and/or fibrosisin a subject that has had GFS. In some instances, the subject isadministered the CD14 antagonist antibody 1, 2, 3, 4, 5, 6 or morehours, 1, 2, 3, 4, 5, 6 or more days, and/or 1, 2, 3, 4, 5, 6 or moreweeks, after the subject has had GFS.

The CD14 antagonist antibody, and optionally the ancillary agent(collectively referred to herein as “therapeutic agents”), will beadministered in an “effective amount(s)”, to achieve an intended purposein a subject, such as the reduction or prevention of one or moresymptoms or consequences of the ocular disease, e.g. the inhibition oramelioration of ocular inflammation, neovascularization and/or fibrosis,inhibition of the development of lesions, and/or inhibition or slowingof loss of vision. The dose of therapeutic agents(s) administered to apatient should be sufficient to achieve a beneficial response in thesubject. In some examples, the administration of the antibody(optionally with an ancillary agent) results in the inhibition oramelioration of ocular inflammation, neovascularization and/or fibrosiscompared to when the antibody is not administered. In other examples,the administration of the antibody (optionally with an ancillary agent)results in a reduction, inhibition or a slowing of a loss of visioncompared to when the antibody is not administered.

In particular examples, the subject with the ocular disease has, or isat risk of developing, ocular fibrosis (e.g. retinal, subretinal orepiretinal fibrosis, including epiretinal membrane (ERM). Thus, in someembodiments, subjects that have, or are at risk of developing, ocularfibrosis are identified or selected for treatment in accordance with themethods of the present disclosure.

For example, in some embodiments, the subject has received an anti-VEGFtherapy for at least 3, 6, 12, 14, 16 or 18 months. As has beenpreviously described, patients with ocular disease (e.g. AMD,pathological myopia (e.g. with CNV), diabetic retinopathy, retinopathyof prematurity, etc.) that are being treated with an anti-VEGF therapycan develop ocular fibrosis (see Daniel et al., 2018, Ophthalmology125(7): 1037-1046; Ahn et al., 2016, Retina, 36(11):2140-2149; Xiao etal., 2020, Br J Ophthalmol., Osaadon, 2014, Eye (Lond), 28(5): 510-520;Li et al., 2015, Int J Ophthalmol. 8(6): 1202-1206; Hu et al., 2017,Journal of Ophthalmology, Article ID 5078565; Tong et al., BMCOphthalmology, 18:150), which itself can be associated with acceleratedloss of vision. Accordingly, in some embodiments the present disclosureprovides methods for treating an ocular disease in a subject byadministering to the subject a CD14 antagonist antibody, and optionallyadministering an ancillary agent or performing an intervention, whereinthe subject has received an anti-VEGF therapy for at least 3, 6, 12, 14,16 or 18 months. The present disclosure also provides methods forinhibiting ocular fibrosis in a subject with an ocular disease, byadministering to the subject a CD14 antagonist antibody, and optionallyadministering an ancillary agent or performing an intervention, whereinthe subject has received an anti-VEGF therapy for at least 12, 14, 16 or18 months. In these embodiments, the subject may have received regular(i.e. at defined or pre-therapy intervals, such as monthly) anti-VEGFtherapy, or intermittent (e.g. “as needed”) anti-VEGF treatment asdetermined by a medical practitioner.

In further examples, the present disclosure provides methods fortreating an ocular disease in a subject by administering to the subjecta CD14 antagonist antibody, and optionally administering an ancillaryagent or performing an intervention, wherein the subject has received atreatment for ocular neovascularization (including CNV) for at least 3,6, 12, 14, 16 or 18 months. Treatment for neovascularization mayinclude, in additions to anti-VEGF agents, tyrosine kinase inhibitorssuch as Tie2 inhibitors, and Ang-2 inhibitors. The present disclosurealso provides methods for inhibiting ocular fibrosis, neovascularizationand/or inflammation in a subject with an ocular disease, byadministering to the subject a CD14 antagonist antibody and optionallyadministering an ancillary agent or performing an intervention, whereinthe subject has received a treatment for ocular neovascularization forat least 3, 6, 12, 14, 16 or 18 months. In these embodiments, thesubject may have received regular (i.e. at defined or pre-determinedintervals, such as monthly) treatment for ocular neovascularization, orintermittent treatment for ocular neovascularization as determined by amedical practitioner (e.g. “as needed”).

Evidence of ocular fibrosis in a subject with ocular disease can also beused to identify or select a patient population for treatment with aCD14 antagonist antibody in accordance with the present disclosure. Forexample, hyperreflective material (HRM) as observed by optical coherencetomography (OCT) is considered a biomarker of fibrosis (see e.g.Casalino et al. Exp Rev Opthamology 15(2):83-91). In other examples, thepresence of fibrotic lesions (or scars) is detected by, for example,fluorescein angiography, color fundus photographs (CFP) or fundusautofluorescence (FAF) imaging, as is known to those skilled in the art(see e.g. Daniel et al., 2014, Ophthalmology, 121(3): 656-666).

In further examples, the subject with an ocular disease is selected fortreatment with an anti-CD14 antagonist antibody on the basis that theyare non-responders to treatment with an anti-VEGF agent. Accordingly, insome embodiments the present disclosure provides methods for treating anocular disease in a subject by administering to the subject a CD14antagonist antibody, and optionally administering an ancillary agent orperforming an intervention, wherein the subject is a non-responder totreatment with anti-VEGF agent. The present disclosure also providesmethods for inhibiting ocular inflammation, neovascularization and/orfibrosis in a subject with an ocular disease, by administering to thesubject a CD14 antagonist antibody, and optionally administering anancillary agent or performing an intervention, wherein the subject is anon-responder to treatment with an anti-VEGF agent. Subjects can becategorized as non-responders to anti-VEGF therapy using standardcriteria known to those skilled in the art. For example, a subject maybe categorized a non-responder if they exhibit no gain (e.g. no changeor a loss (e.g. of more than 0.2 or 5 letters) in BVCA after a period(e.g. 3, 6 or 12 months) of anti-VEGF therapy. In other examples,biomarkers are used to categorize a subject as a non-responder.

The quantity or dose frequency of the therapeutic agent(s) to beadministered may depend on the subject to be treated, inclusive of theirdiagnosis (e.g. the type of ocular disease or the type or severitysymptoms they present with), age, sex, weight and general healthcondition thereof. In this regard, precise amounts of the therapeuticagent(s) for administration will depend on the judgment of thepractitioner. One skilled in the art would be able, by routineexperimentation, to determine an effective, non-toxic amount of a CD14antagonist antibody, and optionally an ancillary agent described herein,for administration to a subject. In particular examples, the amount ofCD14 antagonist antibody administered to a subject is between 0.1 mg/kgand 50 mg/kg, between 0.5 mg/kg and 40 mg/kg, between 2 mg/kg and 20mg/kg or between 5 mg/kg and 10 mg/kg. In particular examples, theamount of CD14 antagonist antibody administered to a subject is (or isabout) 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg/kg.

The CD14 antagonist antibody may be administered to the subject as asingle dose or multiple doses. In particular embodiments, the CD14antagonist antibody is administered as multiple doses. In embodimentswhere the CD14 antagonist antibody is administered as multiple doses,doses may be administered as regular or semi-regular doses (e.g.monthly), or “as-needed”, as determined by a medical practitioner. Insome examples, the CD14 antagonist antibody is administered every 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks, and for a duration of 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 24, 48 or more months or 3, 4, 5, 6, 7, 8, 9 10or more years.

Administration may be any route suitable, including local ocularadministration (e.g. intravitreal, subconjunctival, subretinal,retrobulbar or intracameral administration, including discrete injectionor implantation), topical administration (e.g. by eye drops) or systemicadministration.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting example.

EXAMPLES Example 1 Assessment of the Effect of Anti-CD14 Treatment onCNV

A study was performed to evaluate the effect of an anti-CD14 antagonistantibody in a laser-induced mouse model of choroidal neovascularization(CNV). The active agent used in the study was the biG53 F(Ab′)2antibody, which functionally inhibits PAMP-dependent cytokine productionin a dose dependent manner similar to that observed with the anti-humanCD14 mAb IC14 (International patent application no. PCT/US2020/043619).The effect of this CD14 antagonist was compared to aflibercept (VEGFTrap-eye).

A. Materials and Methods Laser Induced Choroidal Neovascularization

The laser induced mouse model of CNV is a well-established model forevaluating ocular diseases such as wet AMD. Briefly, mice were deeplyanaesthetized by an intraperitoneal (IP) injection of a combination ofketamine (60 mg/kg; Provet, Heatherton, VIC, Australia) and xylazine (13mg/kg; Troy Laboratories, Glendenning, NSW, Australia) and their eyesfurther anaesthetized with 0.5% proparacaine hydrochloride (Alcaine;Alcon Laboratories, Frenchs Forest, NSW, Australia). Pupils were dilatedby topical application of 1% atropine sulfate (Alcon Laboratories) and2.5% phenylephrine hydrochloride (Bausch & Lomb, Chatswood, NSW,Australia). Each animal had four laser burns applied to each eye usingan image guided (Micron IV, Phoenix Research Laboratories, Pleasanton,CA, USA) argon laser (532 nm diode, 350 mW, 70 ms pulse duration).Immediately after laser treatment a fundus image was collected, andocular treatment applied as detailed below.

Intravitreal Injection (IV) of Agents

Treatment concentrations and injections of various agents were asfollows:

-   -   1 μl aliquot of anti-CD14 at 740 μg/ml    -   1.5 and 2 μl aflibercept (15 μg/μl, 60 μg/μl, 80 μg/μl per eye),        prepared by diluting human aflibercept (40 mg/ml) to the        appropriate concentration.    -   1.5 μl anti-VEGF164 neutralizing antibody (200 ng/μl; AF493-NA,        R&D Systems)    -   1 μl PBS

Injections were applied to left and right eyes using a custom Hamiltonsyringe attached to a sterile 31 g needle. Following intravitrealinjection, animals were allowed to recover and placed in their boxes for7 days. A second cohort of animals received 1 μl anti-CD14 or PBS and 2μl aflibercept (80 μg/μl per eye) seven days after laser treatment andthen underwent experimentation at day 14.

Fluorescein Angiography and Tissue Collection

7 days or 14 days after laser treatment animals were anaesthetized witha combination of xylazine and ketamine and fundus fluoresceinangiography was performed on both eyes using a Micron IV retinal camera(Phoenix Research Laboratories, Pleasanton, CA, USA). Images werecollected for each eye immediately after injection of 1% sodiumfluorescein and then and 10 minutes after injection. Following imaging,animals were killed by cervical dislocation, their eyes were removed andplaced in either: (a) fixative containing 60% Ethanol, 5% acetic acid,4% paraformaldehyde, 3% sucrose in distilled water and fixed overnightor (b) the retina and RPE were separated and placed in RLT buffer forRNA analysis.

Analysis of Lesion Size

Following fluorescein angiography, each tiff file was extracted, and thearea of each lesion was quantified using a semi-automated macro in ImageJ (a Java-based image processing program developed at the NationalInstitutes of Health and the Laboratory for Optical and ComputationalInstrumentation. A set of criteria was used for this analysis where alllaser spots with hemorrhage as well as fused lesions were excluded fromanalysis.

Quantification of Lesion Height and Fibrosis

Following sectioning of eye cups, slides were labelled with Masson'strichrome stain and digital images of each lesion were captured. Lesionsize is quantified by measuring the height of the lesion normalizing tothe thickness of the choroid. For each sample, three sections wereanalysed and averaged to obtain a single value.

Total RNA Extraction and Quantitative Real-Time PCR

Quantification of angiogenic and fibrosis gene expression was performedas previously described (Jobling et al. 2015, FASEB J 29(2): 696-710).Total RNA was isolated from frozen RPE/choroidal complexes after 7 and14 days of CNV using commercial spin columns (RNeasy® Micro Kit, Qiagen)incorporating an on-column DNase I digest to remove genomiccontamination. Unlasered tissues from aged matched control mice wereused as controls. RNA concentration and quality were assessed theAgilent 2200 Tape Station (Agilent Technologies 5067-5583 D1000,Germany) according to manufacturer's instructions. Samples with RNAintegrity number (RIN) above 8 were used for quantitative PCR.

For standard real time qPCR, reverse transcription reactions wereperformed on total RNA using random hexamer primers (Omni script,Qiagen) and subsequently diluted to ˜3 ng/μl. Expression of severalgenes related to CNV/fibrosis were assessed relative to the housekeepinggenes, hypoxanthine guanine phophoribosyl transferase (Hprt) andglyceraldehyde-3-phosphate dehydrogenase (Gapdh). External standardswere produced using the same primer pairs that incorporated T7 promoterand polyT15 sequences at the 5′ end of each forward and reverse primer,respectively. The amplified standards were transcribed into copy RNA(Megascript T7 High Yield Transcription kit; Ambion Inc., Austin, TX)and dilutions combined with yeast t-RNA (75 ng; Invitrogen, Carlsbad,CA) to reflect the retinal total RNA amount (75 ng) used in the reversetranscription reaction. The RNA standards were reverse transcribed withthe retinal RNA samples to standardise efficiency.

Real-time PCR was performed on the Rotorgene V3000 (Corbett Research,Australia) using a commercial reaction mixture incorporating SYBR green(SensiFast, Bioline). Respective four-point standard curves wereincluded in every run and standards and samples were amplified intriplicate. Absolute gene copy number was calculated with reference tothe standard curve (Rotorgene V6.1 software, Australia) and expressedrelative to Hprt and Gapdh.

To analyse global changes in gene expression, Fibrosis RT2 Profiler™ PCRArray (Cat #: PAMM-120ZE; Qiagen, Australia) was used. RNA samples fromeach experimental group were pooled (n=9 for each experimental group andn=3 pooled samples). 75 ng of total RNA was amplified, and reversetranscribed into complementary DNA (cDNA) using RT2 PreAMP cDNASynthesis Kit (Qiagen, VIC, Australia) according to the manufacturer'sinstructions (RT2 PreAMP cDNA Synthesis Handbook; Qiagen). Subsequently,RT2 SYBR Green Mastermix (Qiagen) was mixed with each cDNA sample andthen added across the 384-well plate containing 4 replicates of 96-wellassays including 84 fibrosis-related (Table 1), genes, 5 housekeepercontrol genes, 1 genomic DNA control, 3 reverse transcription controlsand 3 positive PCR control.

TABLE 1 Fibrosis-associated genes in PCR array Type of gene GenePro-Fibrotic: Acta2 (a-SMA), Agt, Ccl11 (eotaxin), Ccl12 (MCP-5,Scya12), Ccl3 (Mip-1a), Ctgf, Grem1, Il13, Il13ra2, Il4, 115, Snai1(Snail). Anti-Fibrotic: Bmp7, Hgf, Ifng, Il10, Il13ra2. ExtracellularMatrix Col1a2, Col3a1. (ECM) Structural Constituents: ExtracellularMatrix Lox, Mmp1a, Mmp13, Mmp14, Mmp2, Mmp3, Mmp8, Mmp9, (ECM)Remodeling Plat (tPA), Plau (uPA), Plg, Serpinala, Serpine1 (PAI-1),Enzymes: Serpinh1 (Hsp47), Timp1, Timp2, Timp3, Timp4. Cell AdhesionMolecules: Itga1, Itga2, Itga3, Itgav, Itgb1, Itgb3, Itgb5, Itgb6,Itgb8. Inflammatory Cytokines Ccl11 (eotaxin), Ccl12 (MCP-5, Scya12),Ccl3 (Mip-1a), Ccr2, & Chemokines: Cxcr4, Ifng, Il10, Il13, Il13ra2,Il1a, Il1b Il4, I15, Ilk, Tnf. Growth Factors: Agt, Ctgf, Edn1, Egf,Hgf, Pdgfa, Pdgfb, Vegfa. TGFB Superfamily Bmp7, Cav1, Dcn, Eng (Evi-1),Grem1, Inhbe, Ltbp1, Smad2 Members: (Madh2), Smad3 (Madh3), Smad4(Madh4), Smad6, Smad7, Tgfb1, Tgfb2, Tgfb3, Tgfbr1 (ALK5), Tgfbr2,Tgif1, Thbs1 (TSP-1), Thbs2. Transcription Factors: Cebpb, Jun, Myc,Nfkb1, Sp1, Stat1, Stat6. Epithelial-to- Akt1, Bmp7, Col1a2, Col3a1,Itgav, Itgb1, Mmp2, Mmp3, Mesenchymal Transition Mmp9, Serpine1 (PAI-1),Smad2 (Madh2), Snai1 (Snail), (EMT): Tgfb1, Tgfb2, Tgfb3, Timp1. OtherFibrosis Genes: Bcl2, Fasl (Tnfsf6).

The cDNA was amplified using the Applied Biosystems ViiA 7 Real-Time PCRSystem (Thermo Fisher Scientific, Wilmington, DE, USA) at 95° C. for 15min., followed by 95° C. for 15 seconds and 60° C. for 1 min. (40cycles). To obtain statistically valid data, three independent arrayswere run for each experimental group. The data were analyzed using GeneGlobal RT2 Profiler PCR Data Analysis tool from QIAGEN website.

Quantification of VEGF Protein Expression

At 2, 7, and 14 days after laser treatment, eyes were enucleated, retinaand RPE/choroid isolated and immediately snap frozen in liquid nitrogen.To prepare tissue homogenates, samples were placed in 250 ul of 1×PBSwith added 1% Protease inhibitor cocktail (P8340, Sigma) and sonicatedfor 5 minutes on ice. After performing three freeze-thaw cycles to breakthe cell membranes, the homogenates were placed on ice for 30 minutes,centrifuged at 14,000 rpm for 10 minutes at 4° C. and supernatants werecollected. Total protein concentration was quantified using Pierce™ BCAProtein Assay Kit (ThermoFisher scientific, Australia). VEGF levels wereanalysed by a commercially available ELISA kit (Quantikine; R&D Systems)according to the manufacturer's instructions. The VEGF concentration(pg/ml) in each sample was calculated from the standard curve andnormalized to total protein concentration (pg/mg of total protein). Foreach sample, tissues from both eyes were pooled to obtain sufficientprotein.

Analysis of Innate Immune Cell Migration by Flow Cytometry

Laser-treated eyes were enucleated at day 2 and 7 and RPE/choroidharvested from both eyes. The isolated RPE/choroid was cut in smallpieces, incubated in 5000 of digestion cocktail (0.1 mg/ml Liberase TLand 0.2 mg/ml DNase1) at 37° C. for 60 minutes with mechanicaldissociation at time 20, 40, and 60 minutes. Cells were filtered through40 um nylon strainer, spun at 350 g for 10 minutes and washed twice inFACS buffer containing 1% BSA and 0.1% sodium azide. Cells wereincubated with Fc-Block (553142, BD Biosciences) for 15 minutes, washedtwice and stained with titrated concentrations offluorochrome-conjugated antibodies for 30 minutes. Spleen tissue wasused as a positive control for each run. Antibodies utilised areprovided in Table 2.

Briefly, cells were gated based on CD45 expression to identifyleucocytes (CD45+/CD34−), and then separated based on CD11b, CD11cexpression (dendritic cells and other myeloid cells) and Ly6C and Ly6Gto identify inflammatory monocytes, resident monocytes/macrophages andneutrophils. The proportions of different subpopulations of leucocyteswere quantified in unlasered (non-CNV), vehicle, aflibercept (80 μg/μl),and anti-CD14 treated eyes.

TABLE 2 Antibodies used in flow cytometry. Antibody Target cell typeSupplier ViaKrome 808 Live/dead cells Beckman Coulter CD45 Leukocytes BDBiosciences CD11b Myeloid cells BD Biosciences CD11c Dendritic cellBiolegend Ly6C Monocytes Biolegend Ly6G Neutrophils Biolegend CD14Monocytes/ Macrophages Biolegend MHCII Dendritic cell BiolegendCirculating fibrocytes CCR2 Monocytes/ macrophages Biolegend Circulatingfibrocytes CD34 Fibrocytes Biolegend

B. Results Effect of Treatment on Lesion Size

The size of lesions induced by laser injury is considered a surrogatemeasure of angiogenesis that is associated with choroidalneovascularization (CNV). The effect that different concentrations ofaflibercept or a mouse neutralizing antibody targeting VEGF had inreducing lesion size 7 days after laser induced CNV was assessed. Asshown in FIG. 1A, treatment with aflibercept reduced the size of laserinduced lesions, but only when the dose injected was ˜20 times higherthan what is conventionally used in the treatment of human wet AMD. Incomparison to PBS (vehicle injection), high dose aflibercept (80 μg/μl)significantly reduced the size of lesions 7 days following laser inducedCNV (One-way ANOVA, post-hoc Tukey's test, p<0.001; vehicle vsaflibercept p=0.0048). Fluorescein angiography lesion size followingtreatment with aflibercept (15 μg/μl, 60 μg/μl) and also anti-VEGF164(300 ng/ul) was also compared and no change from vehicle was found(One-way ANOVA, post-hoc Tukey's test).

To put this result in greater context of human disease—those with wetAMD are treated with an 50 μl injection of 40 mg/ml aflibercept into thehuman vitreous which has a volume of 4.4 ml. This equates to a vitrealconcentration of aflibercept of 0.45 mg/ml (or 0.45 μg/μl). 15 μg/μl, 60μg/μl and 80 μg/μl was injected into the mouse eye, which has a vitrealvolume of 8 μl. This equates to vitreal concentrations of aflibercept1.875 μg/μl, 7.5 μg/μl and 10 μg/μl, all far higher than used fortreating human disease.

The efficacy of CD14 treatment with vehicle and high dose aflibercept(80 μg/μl) was then compared. As shown in FIG. 1B, anti-CD14 was aseffective in reducing laser induced lesion size as high doseaflibercept. Notably, anti-CD14 reduced lesion size by (˜34%+4%) whichwas similar to the effect induced by high dose aflibercept (˜32%+8%).

Effect of Treatment on Fibrosis

In order to assess the development of fibrosis following laser inducedCNV, a Masson's trichrome stain was used as a means of labelingcollagen, and then the height of the laser induced lesion relative to anadjacent area of choroid was quantified. Anti-CD14, vehicle, threeconcentrations of aflibercept and anti-VEGF164 on fibrosis 7 days afterlaser induced CNV was compared. As shown in FIG. 2 , anti-CD14significantly reduced fibrosis when compared to the vehicle treatmentand high dose aflibercept (One way ANOVA, posthoc Tukeys test; anti-CD14vs vehicle, p=0.015; anti-CD14 vs aflibercept, p=0.0034; aflibercept vsvehicle p=0.86). The correlation between laser induced lesion size anddevelopment of fibrosis following each of the treatments was thenassessed (FIG. 3 ). As expected, there was a positive correlationbetween increasing lesion size and increasing fibrosis in eyes treatedwith a vehicle (PBS; Pearson's correlation). In contrast, treatment withanti-CD14 showed no correlation, wherein irrespective of lesion size(i.e. angiogenesis), development of fibrosis was uniform.

Quantification of Gene Expression in Eyes Seven Days after LaserTreatment

To further examine the effect of treatments on the development ofangiogenesis and/or fibrosis, quantitative PCR on RPE isolated from eyestreated with vehicle, anti-CD14 or high dose aflibercept was performedseven days after laser treatment. Genes examined included Vegf120,Vegf164, Vegf188, tgfb1, and Col1a1.

Laser induced choroidal neovascularization did not alter the expressionof housekeeping genes, Hprt and Gapdh (data not shown). Quantificationof gene expression compared to either Hprt and Gapdh revealed no changein expression when comparing anti-CD14 with vehicle for any geneexamined (FIGS. 4 and 5 ). As shown in FIG. 5 , high dose afliberceptinduced an increase in expression in VEGF isoforms as well as Co1a1.Tgfb1 expression was not affected by any of the treatments examined.

Analysis using a defined PCR array was used to quantify changes inexpression of 84 genes known to be involved in fibrosis. It was observedthat aflibercept and anti-CD14 have distinct effects on RPE geneexpression. For example, aflibercept treatment increased expression offive genes that are important in the formation of extracellular matrix:Col1a2, 114, Itga2, Mmp2 and Mmp3, something not seen with theadministration of the anti-CD14 antibody.

Effect of treatment of expression of VEGF in RPE

In view of the changes in fluorescein lesion size, it was next examinedwhether anti-CD14 reduced expression of VEGF in the RPE compared tovehicle and aflibercept. As shown in FIG. 6 , VEGF protein expressionwas far higher in the RPE than retina. VEGF expression was reduced byanti-CD14 compared to vehicle controls 2 and 7 days after laser inducedCNV in the RPE (Two-way ANOVA, day-ns; treatment-ns; interactionp=0.014; posthoc Tukeys test p=0.02). These results are consistent withthe reduction in laser induced lesion size shown above in FIG. 1 andsuggest that anti-CD14 has effects on VEGF expression.

Effect of Treatment on Recruitment of Innate Immune Cell Populations

An important mechanism proposed to contribute to the development offibrosis is through the effect that influx of innate immune cells intothe subretinal space has on the RPE. Flow cytometry on isolatedRPE/choroid complexes was used to ascertain whether anti-CD14 and/oraflibercept influenced the recruitment of innate immune cell populationsinto the posterior eye.

FIG. 7 shows the percentage of single live cells in the RPE/choroid thatare leucocytes (CD45+), myeloid cells, dendritic cells, inflammatorymonocytes, resident monocytes/macrophages, and neutrophils presentwithin RPE/choroids 2 days after laser induced CNV. Overall, there wasan increase in recruitment into the posterior eye of all six cell types,and treatment with aflibercept or anti-CD14 did not alter thisrecruitment. It was also confirmed whether different proportions ofcells that make of the broad group of leucocytes showed any changes,i.e. the change if any in the proportion of CD45+ leucocytes that ismyeloid cells, dendritic cells, inflammatory monocytes,monocytes/macrophages or neutrophils. In all cell types, whilst therewas an increase in the proportion within the make-up of CD45+leucocytes, this was unaffected by treatment with either aflibercept oranti-CD14 (data not shown).

FIG. 8 shows the recruitment of leucocytes, and the proportion ofleucocytes that is represented by myeloid cells, dendritic cells,inflammatory monocytes, resident monocytes/macrophages, and neutrophilspresent within RPE/choroids 7 days after laser induced CNV. The mostsignificant trend observed at this time point was a reduction inCD45+/CD11b⁺/Ly6G⁺ neutrophils in eyes treated with anti-CD14.

A comparison in the recruitment of leucocytes and myeloid cells between2 and 7 days is shown in FIG. 9 . Importantly, leucocytes andCD11b⁺/CD11c⁻ myeloid cells show greater recruitment 2 days after laserinduced CNV compared to 7 days. Moreover, anti-CD14 treatment reducedthe number of neutrophils compared to vehicle controls 7 days afterlaser induced CNV. These findings suggest that there is an immediateinflux of a range of leucocyte and cells in response to laser, althoughanti-CD14 may have a role in reducing neutrophil numbers after 7 days.

The potential role of aflibercept and anti-CD14 treatment in modifyingrecruitment of circulating fibrocytes (CD45⁺/CD34⁺) into the posterioreye was examined by gating for CCR2 and MHCII (CCR2⁻/MHCII⁻). Two daysafter laser induced CNV, there was an increase in recruitment ofCD45+/CD34+ fibrocytes (FIG. 10 ). However, neither aflibercept noranti-CD14 influenced recruitment of fibrocyte at this time. However,after 7 days, aflibercept showed an increase in recruitment, that wasnot observed in eyes treated with anti-CD14.

The next analysis performed was to assess the expression of CD14 on thesurface of myeloid cells. As shown in FIG. 11 , laser induced CNV wasfound to increase the recruitment of CD14+ myeloid cells by two days andwas also associated with an increase in surface expression of CD14amongst inflammatory monocytes, resident monocytes/macrophages, anddendritic cells. Treatment with anti-CD14 abrogated the laser inducedchange in CD14 expression in dendritic cells.

Laser induced effects on CD14+ recruitment and expression 7 days afterlaser treatment are shown in FIG. 12 . There was an increase inrecruitment of CD14⁺ myeloid cells by seven days. CD14 expression wasincreased after laser induced CNV only in neutrophils. Treatment withaflibercept or anti-CD14 had no effect on these changes in CD14recruitment and expression.

FIG. 13 shows the laser induced effects on CD14 recruitment andexpression over 2 and 7 days. Notably, an increase in CD14 expressionwas noted on inflammatory monocytes after 2 days, that was restored tobaseline by 7 days. However, treatment with aflibercept or anti-CD14 hadno effect in influencing CD14 recruitment or expression.

C. Discussion

This study clearly demonstrates that administration of an anti-CD14antagonist antibody reduced both laser-induced lesion size and fibrosisat 7 days, and significantly reduced the level of VEGF proteinexpression in the RPE between 2 and 7 days after laser-induced CNV(compared to vehicle-treated CNV). By comparison, aflibercept reducedlaser-induced lesion size but not fibrosis at 7 days and this wasassociated with an increased gene expression of all VEGF isoforms andCol1a1 in the RPE at 7 days (although this increase in VEGF geneexpression may be a compensatory mechanism to use of high dose (80μg/μl) of aflibercept).

In order to compare the effect of anti-CD14 with standard treatment, theeffect of three doses of aflibercept as well as a mouse anti-VEGF164neutralizing antibody on fluorescein leakage and fibrosis was examined.In contrast to the dosage of aflibercept used in clinical practice fortreatment of wet AMD (dosage in the eye ˜0.45 mg/ml or 0.45 μg/μl), onlya 20 fold higher dose was found to be effective in the mouse induced CNVmodel (the 80 μg/μl injection results in vitreal concentration of 10μg/μl). This may be because of differences in specificity of afliberceptfor mouse VEGF compared to human VEGF.

It was observed that high dose aflibercept reduced lesion size followinglaser treatment but did not reduce fibrosis. This is consistent withclinical results that show with long term treatment using aflibercept,fibrosis reduces vision in approximately 50% of patients with wet AMD.The studies show that in vehicle treated (and untreated) eyes, there isa correlation between fluorescein lesion size and fibrosis. When lesionsfail to heal (i.e. are larger), the level of fibrosis is greater. Onthis basis, a treatment (e.g. aflibercept) that reduces lesion size inthis mouse model might be expected to reduce fibrosis and any comparisonof treatments such as between aflibercept and anti-CD14 might beexpected to reduce lesion size and fibrosis simply by different extents.However, this was not the case for any concentration ofaflibercept—neither aflibercept nor anti-VEGF164 reduced fibrosis to anyextent. In contrast, treatment with anti-CD14 did reduce fibrosis andimportantly, did this in a manner that was independent of lesion size.

In order to further study whether the anti-CD14 antibody attenuatedrecruitment of leucocytes especially myeloid cells into the posterioreye, the recruitment of different types of leucocytes and myeloid cellsin the RPE/choroid in non-lasered control, PBS treated CNV, as well asanti-CD14 and aflibercept treated eyes, was assessed. Althoughrecruitment of leucocytes and various myeloid cells was found to beincreased within 2 days of laser induced CNV, neither anti-CD14 antibodynor aflibercept had a substantial effect in reducing recruitment ofcells. The results that laser-induced CNV induced recruitment of amyeloid cells is consistent with previous studies (Tsutsumi et al. 2003,J Leukoc Biol 74(1): 25-32; Droho et al. 2019, Invest Ophthalmol Vis Sci60(15): 5059-5069). The observation that neither aflibercept noranti-CD14 antibody appeared to alter the broad recruitment of myeloidcells is most likely explained by the heterogeneity of cell classesevaluated. It is possible that aflibercept or anti-CD14 antibody,targets a highly specific cell type that represents only a smallfraction of the cell classes examined.

To address the possibility that aflibercept or anti-CD14 antibodytargets a single subtype of myeloid cells, the effect on the recruitmentof CD34+CCR2+MHCII+ fibrocytes was investigated. It was observed that,like other myeloid cells, there was enhanced recruitment by two daysafter laser injury. There was a trend for a reduction in this cellpopulation in anti-CD14 antibody treated posterior eyes compared toaflibercept treated eyes after 7 days.

The reduction in protein levels of VEGF in the RPE but not the retina,and in the absence of an increase in gene expression, followinganti-CD14 treatment was of particular interest. The localized reductionof VEGF in the RPE rather than the retina indicated that it is likelynot from infiltrating inflammatory cells (e.g. a reduction in VEGFproduction due to anti-CD14 attenuation of macrophage activities), asthese inflammatory cells would be present in the retina rather than RPE.Rather, and quite unexpectedly, it appears that the reduction in VEGF inthe RPE is the result of antagonism of the mCD14 receptors expressed onthe RPE. This further indicates that the unexpected anti-fibroticactivity of the anti-CD14 antibody following laser-induced CNV isindependent of, and distinct from, its anti-inflammatory andanti-angiogenic (i.e. anti-neovascularisation) activity.

In conclusion, it is clear that administration of an anti-CD14 antibodyreduces both lesion size and fibrosis resulting from laser-induced CNV,and does this in a manner that is in the absence of any overt oculartoxicity. Interestingly, the reduction in fibrosis in the retina appearsto be through a novel mechanism that is not related to lesion size (i.e.the degree of neovascularisation): inhibition of fibrosis followingtreatment with anti-CD14 was uniform and irrespective of the inhibitionof lesion size, and VEGF proteins levels were reduced in the RPE but notthe retina, suggesting that this was through antagonism of the mCD14receptors expressed on the RPE rather and not anti-CD14 attenuation ofinfiltrating macrophage activity. Thus, it is clear that the anti-CD14antibody acts on the posterior eye in a manner that is distinct from,and is beneficial in comparison to, aflibercept, which was found not toreduce fibrosis and indeed was observed to increase expression of genesthat are important in the formation of extracellular matrix.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims.

1.-59. (canceled)
 60. A method for reducing ocular fibrosis in a subjectwith wet age-related macular degeneration (AMD) and ocular fibrosis,comprising administering to the eye of the subject an amount of a CD14antagonist antibody effective to reduce ocular fibrosis in the subject,wherein the CD14 antagonist antibody is either administered alone or incombination with an anti-VEGF agent.
 61. The method of claim 60, whereinthe CD14 antagonist antibody comprises: a) an antibody VL domain, orantigen binding fragment thereof, comprising L-CDR1, L-CDR2 and L-CDR3,wherein L-CDR1 comprises the sequence RASESVDSYVNSFLH [SEQ ID NO: 13],L-CDR2 comprises the sequence RASNLQS [SEQ ID NO: 14], and L-CDR3comprises the sequence QQSNEDPYT [SEQ ID NO: 27]; and b) an antibody VHdomain, or antigen binding fragment thereof, comprising H-CDR1, H-CDR2and H-CDR3, wherein H-CDR1 comprises the sequence SDSAWN [SEQ ID NO:16], H-CDR2 comprises the sequence YISYSGSTSYNPSLKS [SEQ ID NO: 17], andH-CDR3 comprises the sequence GLRFAY [SEQ ID NO: 18].
 62. The method ofclaim 60, wherein the CD14 antagonist antibody comprises: a light chaincomprising the amino acid sequence: [SEQ ID NO: 28]QSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 

 and a heavy chain comprising the amino acid sequence: [SEQ ID NO: 29]LQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

 or a light chain comprising the amino acid sequence: [SEQ ID NO: 32]DIVLTQSPASLAVSLGQRATISCRASESVDSYVNSFLHWYQQKPGQPPKLLIYRASNLQSGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC

 and a heavy chain comprising the amino acid sequence: [SEQ ID NO: 33]DVQLQQSGPGLVKPSQSLSLTCTVTGYSITSDSAWNWIRQFPGNRLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCVRGLRFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K.


63. The method of claim 60, wherein the CD14 antagonist antibody is ofthe IgG1 isotype.
 64. The method of claim 60, wherein the CD14antagonist antibody is of the IgG1 isotype and comprises L234A and L235Amutations.
 65. The method of claim 60, wherein the CD14 antagonistantibody is a chimeric, humanized or human antibody.
 66. The method ofclaim 60, wherein the subject has received an anti-VEGF agent for atleast 3, 6, 9, 12, 14, 16 or 18 months.
 67. The method of claim 60,wherein the anti-VEGF agent is selected from the group consisting of ananti-VEGF antibody, anti-VEGF antibody mimetic, VEGF Trap molecule,soluble fms-like tyrosine kinase-1, or a tyrosine kinase inhibitor. 68.The method of claim 67, wherein the anti-VEGF antibody is selected fromthe group consisting of brolicizumab, ranibizumab and faricimab.
 69. Themethod of claim 67, wherein the VEGF Trap molecule is aflibercept orconbercept.
 70. The method of claim 67, wherein the anti-VEGF antibodymimetic is abicipar pegol.
 71. The method of claim 67, wherein thetyrosine kinase inhibitor is a Tie2 inhibitor or an Ang-2 inhibitor. 72.The method of claim 60, wherein the CD14 antagonist antibody isadministered in combination with an anti-VEGF agent.
 73. The method ofclaim 72, wherein the anti-VEGF agent is selected from the groupconsisting of an anti-VEGF antibody, anti-VEGF antibody mimetic, VEGFTrap molecule, soluble fms-like tyrosine kinase-1, and a tyrosine kinaseinhibitor.
 74. The method of claim 73, wherein the anti-VEGF antibody isselected from the group consisting of brolicizumab, ranibizumab andfaricimab.
 75. The method of claim 73, wherein the VEGF Trap molecule isaflibercept or conbercept.
 76. The method of claim 73, wherein the VEGFantibody mimetic is abicipar pegol.
 77. The method of claim 73, whereinthe tyrosine kinase inhibitor is a Tie2 inhibitor or an Ang-2 inhibitor.78. The method of claim 72, wherein the subject has received ananti-VEGF agent for at least 3, 6, 9, 12, 14, 16 or 18 months.